Nucleotide and amino acid sequences from Xenorhabdus and uses thereof

ABSTRACT

The invention provides isolated nucleotide sequences from  Xenorhabdus nematophila  species Xs86068, and, in particular, nucleotide sequences that encode insect inhibitory proteins, the insecticidal proteins, and compositions that comprise one or more of the insecticidal proteins for use in controlling insect infestation.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 11/216,782, filed Aug. 31, 2005 now U.S. Pat. No. 7,319,142, whichclaims the benefit under 35 U.S.C. §119(e) to U.S. ProvisionalApplication 60/606,098, filed Aug. 31, 2004. The entirety of each ofthese applications is hereby incorporated by reference, including thesequence listings.

INCORPORATION OF SEQUENCE LISTING

A computer readable form of the sequence listing on CD-R, containing thefile named “52035B_Xeno Seq Listing.txt”, which is 28,006,000 bytes insize (measured in Windows XP) and which was recorded on Aug. 5, 2005 andfiled in U.S. application Ser. No. 11/216,782, is hereby incorporated byreference.

BACKGROUND OF THE INVENTION

The present invention relates to nucleic acid sequences from Xenorhabdusnematophila. The invention also relates to methods of using thedisclosed nucleic acid molecules to encode proteins and fragments ofproteins and to develop antibodies, for example, for nucleic acidsequence identification and analysis, preparation of constructs,transformation of cells such as bacterial cells and plant cells with thenucleotide compositions disclosed herein to produce Xenorhabdus proteinsor fragments thereof, in particular novel insect inhibitory,bactericidal, fungicidal and nematocidal proteins.

Xenorhabdus is a Gram-negative bacterium, a member of the family ofEnterobacteriaceae, and symbiotically associated with nematodes of thegenus Steinernema. The nematode-bacterial complex can be characterizedas an obligate and lethal parasitic relationship, specializing inparasitizing and proliferating in soil insect larvae. Infective,non-feeding stages of these nematodes live in soil and carry in theirgut the nematode-genus-specific symbiotic bacteria. It is believed thatthe nematodes actively search for the appropriate insect host, invadethe insect larvae through natural openings or lesions in the cuticleand, once inside the hemolymph, release their symbiotic bacteria. Thenematode-bacterial complex secretes a variety of highly efficientextracellular metabolites and proteins exhibiting insecticidal,bactericidal, fungicidal and nematocidal properties to secure the larvalmass as a source of nutrition. An array of extracellular enzymes such aslipases, phospholipases, proteases, nucleases as well as several broadspectrum antibiotics, and antifungal and nematocidal compositions arealso secreted [Boemare & Akhurst, J. Gen. Microbiol. 134: 751-761, 1988;Li et al., Can. J. Microbiol. 43(8):770-773, 1997; McInerney et al., J.Nat. Prod. 54(3):774-84, 1991; McInerney et al., J. Nat. Prod.54(3):785-95, 1991; Sundar and Chang, J. Gen. Microbiol. 139 (Pt12):3139-48, 1993]. It has been discovered that some compounds secretedby Xenorhabdus exhibit anti-neoplastic (U.S. Pat. No. 5,827,872),acaricidal, anti-inflammatory and anti-ulcerogenic properties (U.S. Pat.No. 4,837,222). U.S. Pat. No. 6,048,838 describes insect inhibitoryproteins that exhibit a molecular weight of greater than 100 kDaproduced by Xenorhabdus sp., which are active against a variety ofinsect species including the orders, Lepidoptera, Coleoptera, Diptera,and Acarina, when provided in the insect diet.

Xenorhabdus strains have been shown to produce an array of extracellularproteins and small molecules or secondary metabolites having specializedfunctions [Li et al., Can. J. Microbiol. 43(8):770-773, 1997; McInerneyet al., J. Nat. Prod. 54(3):774-84, 1991; U.S. Pat. No. 6,048,838]. Morecommercially interesting are proteins and small molecules havingantibiotic properties or proteins that exhibit insect inhibitoryactivity. A small number of insect inhibitory proteins have previouslybeen identified from these bacteria, symbionts of insect-parasiticnematodes (Morgan et al., Appl. Environ. Microbiol., 67(5):2062-2069,2001; U.S. Pat. No. 6,048,838). Such proteins and compositions are usedas biologically safe and effective pest control agents. Unlike chemicalpesticide compositions, these proteins have no effect upon theenvironment in general, can be targeted to direct their effect primarilyupon target insect species, and have no effect on non-target species.Therefore, a different resistance management strategy that takesadvantage of insect inhibitory proteins derived from distinct microbialsources other than B. thuringiensis would be desirable. Insectinhibitory proteins isolated from Xenorhabdus bacteria seem to have allthe prerequisites for the delivery of novel genes for transgenicexpression of insect pest inhibiting proteins to provide pest resistanceto plants, either alone or in combination with Bacillus thuringiensisinsecticidal proteins.

Therefore, there is a great deal of interest in identifying the genesthat encode new insect inhibiting proteins, and proteins involved in thebiosynthetic pathways of novel antibiotics produced by Xenorhabdusbacteria, as well as other useful proteins. Isolation and sequencing ofthe entire genome of Xenorhabdus would facilitate such an endeavor,because it would allow dissection and analysis of the genome intodiscrete genes encoding proteins having beneficial properties asdescribed herein.

SUMMARY OF THE INVENTION

In one embodiment, the present invention provides isolated nucleotidesequences from Xenorhabdus nematophila bacterium, strain Xs86068. Theisolated nucleotide sequences encode at least a group of insectinhibitory proteins that have insecticidal activities against insectpests. The insect inhibitory proteins of the present inventioncomprises, but are not limited to, the following amino acid sequences:SEQ ID NO:6903, SEQ ID NO:6904, SEQ ID NO:6905, SEQ ID NO:7110, SEQ IDNO:7179, SEQ ID NO:7514, SEQ ID NO:7776, SEQ ID NO:7777, SEQ ID NO:7803,SEQ ID NO:8275, SEQ ID NO:8277, SEQ ID NO:8279, SEQ ID NO:8280, SEQ IDNO:8281, SEQ ID NO:8454, SEQ ID NO:8468, SEQ ID NO:8595, SEQ ID NO:9946,SEQ ID NO:10477, SEQ ID NO:10481, SEQ ID NO: 10482, SEQ ID NO:10483, SEQID NO:10484, SEQ ID NO:10485, SEQ ID NO:10486, SEQ ID NO:10487, SEQ IDNO:10488, SEQ ID NO:10551, SEQ ID NO:11147, SEQ ID NO:11688, SEQ IDNO:11690, SEQ ID NO:11691, SEQ ID NO:11692, and SEQ ID NO:11693. Theinsecticidal activities are manifested by inhibiting the growth ordevelopment of, or contributing substantially to the death of, an insectfrom the insect orders Coleoptera [e.g., western corn rootworm (WCR,Diabrotica virgifera virgifera LeConte), and cotton boll weevil (BWV,Anthomonas grandis grandis), and Hemiptera [e.g., Western TarnishedPlant Bug (WTPB, Lygus hesperus Knight)]. The present insect inhibitoryproteins also have insecticidal activities against insect pests fromother insect orders such as Diptera and Hymenoptera and against suckingand piercing insects or insect larvae.

The isolated nucleotide sequences of the present invention that encodethe insect inhibitory proteins comprise, but are not limited to, thefollowing: SEQ ID NO:795, SEQ ID NO:796, SEQ ID NO:797, SEQ ID NO:1002,SEQ ID NO:1071, SEQ ID NO:1406, SEQ ID NO:1668, SEQ ID NO:1669, SEQ IDNO:1695, SEQ ID NO:2167, SEQ ID NO:2169, SEQ ID NO:2171, SEQ ID NO:2172,SEQ ID NO:2173, SEQ ID NO:2346, SEQ ID NO:2360, SEQ ID NO:2487, SEQ IDNO:3838, SEQ ID NO:4369, SEQ ID NO:4373, SEQ ID NO:4374, SEQ ID NO:4375,SEQ ID NO:4376, SEQ ID NO:4377, SEQ ID NO:4378, SEQ ID NO:4379, SEQ IDNO:4380, SEQ ID NO:4443, SEQ ID NO:5039, SEQ ID NO:5580, SEQ ID NO:5582,SEQ ID NO:5583, SEQ ID NO:5584 and SEQ ID NO:5585.

In another embodiment, the present invention provides purified insectinhibitory proteins from Xenorhabdus nematophila bacterium, strainXs86068, which are active against insect pests. The insect inhibitoryproteins of the present invention at least comprises, but are notlimited to, the following amino acid sequences: SEQ ID NO:6903, SEQ IDNO:6904, SEQ ID NO:6905, SEQ ID NO:7110, SEQ ID NO:7179, SEQ ID NO:7514,SEQ ID NO:7776, SEQ ID NO:7777, SEQ ID NO:7803, SEQ ID NO:8275, SEQ IDNO:8277, SEQ ID NO:8279, SEQ ID NO:8280, SEQ ID NO:8281, SEQ ID NO:8454,SEQ ID NO:8468, SEQ ID NO:8595, SEQ ID NO:9946, SEQ ID NO:10477, SEQ IDNO:10481, SEQ ID NO:10482, SEQ ID NO:10483, SEQ ID NO:10484, SEQ IDNO:10485, SEQ ID NO:10486, SEQ ID NO:10487, SEQ ID NO:10488, SEQ IDNO:10551, SEQ ID NO:11147, SEQ ID NO:11688, SEQ ID NO:11690, SEQ IDNO:11691, SEQ ID NO:11692, and SEQ ID NO:11693.

In still another embodiment, the present invention provides aninsecticidal composition that comprises one or more insect inhibitoryproteins as specified above having an amino acid sequence selected fromthe group consisting of SEQ ID NO:6903, SEQ ID NO:6904, SEQ ID NO:6905,SEQ ID NO:7110, SEQ ID NO:7179, SEQ ID NO:7514, SEQ ID NO:7776, SEQ IDNO:7777, SEQ ID NO:7803, SEQ ID NO:8275, SEQ ID NO:8277, SEQ ID NO:8279,SEQ ID NO:8280, SEQ ID NO:8281, SEQ ID NO:8454, SEQ ID NO:8468, SEQ IDNO:8595, SEQ ID NO:9946, SEQ ID NO:10477, SEQ ID NO:10481, SEQ IDNO:10482, SEQ ID NO:10483, SEQ ID NO:10484, SEQ ID NO:10485, SEQ IDNO:10486, SEQ ID NO:10487, SEQ ID NO:10488, SEQ ID NO:10551, SEQ IDNO:1147, SEQ ID NO:11688, SEQ ID NO:11690, SEQ ID NO:11691, SEQ IDNO:11692, and SEQ ID NO:11693. The insecticide composition disclosedherein may comprise one of the following: insecticidal proteinsuspensions, isolated protein components or bacterial cells that aretransformed with one or more nucleotide sequences that encode theinsecticidal inhibitory proteins of the invention. The insecticidalcomposition may be formulated in the form of a dust, a granularmaterial, an oil (vegetable or mineral) suspension, a water suspension,a mixture of oil and water emulsion, or a wettable powder, or incombination with an agriculturally acceptable carrier that may be solidor liquid.

In a further embodiment, the present invention provides an isolatedXenorhabdus nematophila bacterium, strain Xs86068. The inventors havedemonstrated that this strain exhibits insecticidal activity againstcommercially important insect species including, e.g., those in theorders Coleoptera (e.g., WCR, BWV), and Hemiptera (e.g., WTPB). Thestrain may have insecticidal activity against other insects including,e.g., Dipteran and Hymenopteran insects, or a sucking and piercinginsect or an insect larva thereof. The insect inhibitory proteins alsohave insecticidal activities against insect pests from other insectorders. This strain may be used as a source for DNA sequences encodinginsecticidal proteins, and when formulated into a composition of matteras a spray, powder or emulsion, for the treatment of plants or animalsto inhibit insect infestation. The Xs86068 strain was deposited on Jul.26, 2004, with the Agriculture Research Culture Collection (NRRL)International Depository Authority at 1815 North University Street, inPeoria, Ill. 61604 U.S.A., according to the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedures and was designated as NRRL B-30757.

The present invention relates to a plant cell, a mammalian cell, abacterial cell other than a X. nematophila Xs86068 cell, an algal cell,an insect cell and a fungal cell transformed with an isolated nucleicacid molecule of the present invention that is selected from the groupconsisting of SEQ ID NO:795, SEQ ID NO:796, SEQ ID NO:797, SEQ IDNO:1002, SEQ ID NO:1071, SEQ ID NO:1406, SEQ ID NO:1668, SEQ ID NO:1669,SEQ ID NO:1695, SEQ ID NO:2167, SEQ ID NO:2169, SEQ ID NO:2171, SEQ IDNO:2172, SEQ ID NO:2173, SEQ ID NO:2346, SEQ ID NO:2360, SEQ ID NO:2487,SEQ ID NO:3838, SEQ ID NO:4369, SEQ ID NO:4373, SEQ ID NO:4374, SEQ IDNO:4375, SEQ ID NO:4376, SEQ ID NO:4377, SEQ ID NO:4378, SEQ ID NO:4379,SEQ ID NO:4380, SEQ ID NO:4443, SEQ ID NO:5039, SEQ ID NO:5580, SEQ IDNO:5582, SEQ ID NO:5583, SEQ ID NO:5584, and SEQ ID NO:5585. In an eventwhen a bacterial cell is used, the bacterium may be selected from thegroup consisting of Bacillus, Agrobacterium, Pseudomonas, Rhizobium,Erwinia, Azotobacter, Azospirillum, Klebsiella, Flavobacterium andAlcaligenes.

Both the isolated nucleotide sequences and the amino acid sequences ofthe present invention are provided in the Sequence Listing file inelectronic format that also includes other nucleotide and amino acidsequences, all of which are set forth in SEQ ID NO:1 through SEQ IDNO:16918, the electronic copy being included on the CD-ROM thataccompanies this specification.

Various advantages and features of the present invention will becomehereinafter apparent, and the nature of the invention may be moreclearly understood, by reference to the following detailed descriptionof the embodiments of the invention and to the appended claims.

BRIEF DESCRIPTION OF THE SEQUENCES

All the nucleotide and amino acid sequences as set forth in SEQ ID NO:1through SEQ ID NO:16918 and as categorized below are provided inelectronic format within the Sequence Listing file, the electronic copybeing included on the CD-ROM that accompanies this specification.

SEQ ID NO:1 through SEQ ID NO:6108 are nucleotide sequences isolatedfrom X. nematophila strain Xs86068. Each of these nucleotide sequencesreside within a larger sequence referred to as a contig sequence, andare cross referenced in the SEQ LISTING file with reference to SEQ IDNO:'s corresponding to a particular contig sequence.

SEQ ID NO:6109 through SEQ ID NO:12216 represent amino acid sequencesencoded by the nucleotide sequences as set forth at SEQ ID NO:1 throughSEQ ID NO:6108, respectively. SEQ ID NO:6109 represents the amino acidsequence translation of SEQ ID NO:1, SEQ ID NO:6110 represents the aminoacid sequence translation of SEQ ID NO:2, and so on and so forth.

SEQ ID NO:12217 through SEQ ID NO:14867 represent predicted promoternucleotide sequences isolated from X. nematophila strain Xs86068.

SEQ ID NO:14868 through SEQ ID NO:16342 represent terminator nucleotidesequences isolated from X. nematophila strain Xs86068.

SEQ ID NO:16343 through SEQ ID NO:16424 represent nucleotide sequencesisolated from X. nematophila strain Xs86068 encoding tRNA's and rRNA's.

SEQ ID NO:16425 through SEQ ID NO:16918 represent the contig sequences.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, novel nucleic acid moleculeshave been isolated from a bacterium X. nematophila, strain Xs86068, andtheir encoded polypeptides or proteins are provided. Isolated nucleicacid molecules comprising regulatory elements that include promoter andterminator sequences are also provided. In a preferred embodiment, thepresent invention provides isolated nucleic acid molecules that encodesa class of proteins that exhibits insect inhibitory activity, whereinthe activity is manifested by inhibiting the growth or development of,or contributing substantially to, or causing the death of an insect,such as a Coleopteran, a Dipteran, a Lepidopteran, a Hemipteran, aHymenopteran, or a sucking and piercing insect or insect larvae thereof.Therefore, those skilled in the art will find the utility of theseinsecticidal proteins in protecting plants from insect infestations.

The present invention also provides isolated nucleic acid moleculesencoding other types of useful proteins and compositions, e.g., insectinhibitory proteins, microbial inhibitory proteins includingbactericidal and fungistatic proteins, nematocidal and protein homologsof chitinases, histones and restriction enzymes, proteases, proteinscapable of conferring resistance to heavy metals or other toxiccompositions, polyketide synthases, among others.

The present invention relates to methods of obtaining the disclosednucleic acid molecules and proteins and of using the disclosed nucleicacid molecules, proteins, fragments of proteins, and antibodies, forexample, for gene identification and analysis, preparation ofconstructs, transformation of cells with nucleotide compositionsdisclosed herein to produce Xenorhabdus proteins or fragments thereof,in particular novel insect inhibitory, bactericidal, fungicidal andnematocidal proteins.

A computer-readable form of the sequence listing file is recorded onto acomputer readable media, i.e., a compact disc (CD-ROM), labeled as “Seq.Listing” and accompanies the specification. The CD-ROM was created onAug. 31, 2005. The computer readable form contains the sequence listingfile in text file format that comprises SEQ ID NO:1 through SEQ IDNO:16918 which is about 25.5 megabytes, incorporated herein byreference.

One aspect of the present invention relates to an isolated nucleic acidmolecule having a nucleotide sequence, wherein: (1) the nucleotidesequence hybridizes under stringent conditions to a nucleotide sequenceof a second isolated nucleic acid molecule selected from the groupconsisting of SEQ ID NO:1 through SEQ ID NO:6108 or complements thereof,(2) the nucleotide sequence is a portion of any sequence selected fromthe group consisting of SEQ ID NO:1 through SEQ ID NO:6108; or (3) thenucleotide sequence is the complement of (1) or (2).

The term “an isolated nucleic acid” refers to a nucleic acid that is nolonger accompanied by some of materials with which it is associated inits natural state or to a nucleic acid the structure of which is notidentical to that of any of naturally occurring nucleic acid. Examplesof an isolated nucleic acid include: (1) DNAs which have the sequence ofpart of a naturally occurring genomic DNA molecules, but are not flankedby two coding sequences that flank that part of the molecule in thegenome of the organism in which it naturally occurs; (2) a nucleic acidincorporated into a vector or into the genomic DNA of a prokaryote oreukaryote in a manner such that the resulting molecule is not identicalto any naturally occurring vector or genomic DNA; (3) a separatemolecule such as a cDNA, a genomic fragment, a fragment produced bypolymerase chain reaction (PCR), or a restriction fragment; (4)recombinant DNAs; and (5) synthetic DNAs. An isolated nucleic acid mayalso be comprised of one or more segments of cDNA, genomic DNA orsynthetic DNA.

Stringent conditions are sequence dependent and will be different undervarious circumstances. Generally, stringent conditions are selected tobe about 5° C. lower than the thermal melting point (Tm) for thespecific sequence at a defined ionic strength and pH. The Tm is thetemperature (under defined ionic strength and pH) at which 50% of thetarget sequence hybridizes to a perfectly matched probe. Appropriatestringent conditions are known to those skilled in the art or can befound in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y.6.3.1-6.3.6 (1989). For the purposes of this disclosure, stringentconditions include at least one wash (usually 2) in 0.2×SSC at atemperature of at least about 50° C., usually about 55° C., for 20minutes, or equivalent conditions.

The hybridization portion of the two hybridizing nucleic acids isusually at least 50 nucleotides in length, more usually at least about75 nucleotides in length, more particularly at least 100 nucleotides inlengths. The hybridizing portion of the hybridizing nucleic acid is atleast 70%, at least 80%, at least 90%, or at least 98% identical to thesequence of a portion of a sequence as set forth in SEQ ID NO:1 to SEQID NO:6108.

Another aspect of the present invention relates to an isolated nucleicacid molecule comprising one or more open reading frames as set forth inSEQ ID NO:1 to SEQ ID NO:6108. An “open reading frame” (ORF) is a regionof a nucleotide sequence that encodes a polypeptide. This region mayrepresent a portion of a coding sequence or a total coding sequence.Open reading frames in genomic sequences can be screened for thepresence of protein homologues utilizing one or a number of differentsearch algorithms that have been developed, one example of which are thesuite of programs referred to as BLAST programs. The open reading framesidentified in the isolated nucleic acid molecules comprise SEQ ID NO:1through SEQ ID NO:6108, wherein the open reading frames encodeXenorhabdus proteins or polypeptide or fragments thereof which arehomologues of known proteins or unknown proteins. It has been discoveredthat the nucleic acids and amino acids encoded by the nucleic acidsderived from Xenorhabdus species (bacteria commonly symbioticallyassociated with insect pathogenic nematodes) are surprisingly useful inproviding compositions comprising insect inhibitory proteins, microbialinhibitory proteins including bactericidal, bacteriostatic, fungicidal,and fungistatic proteins, protein homologs of chitinases, histones andrestriction enzymes, proteases, proteins capable of conferringresistance to heavy metals or other toxic compositions, proteins andcompositions capable of conferring pharmaceutical advantages such asantineoplastic, acaricidal, anti-inflammatory and anti-ulcerogenicproperties, polyketide synthases, transposons and mobile geneticelements and their corresponding transposases, excisases, integrases,and invertases, phage and phage particle proteins, other useful proteinshomologous to proteins derived from Xenorhabdus, Photorhabdus, Serratia,Yersinia, Salmonella, E. coli, and Erwinia sp. among others. Inaddition, antibodies directed to the above-mentioned proteins andfragments thereof have been discovered to be of particular utility inthe present invention. The invention also relates to methods of usingthe disclosed nucleic acid molecules, proteins, fragments of proteins,and antibodies, for example, for nucleotide sequence identification andanalysis, preparation of constructs, transformation of cells withnucleotide compositions disclosed herein to produce Xenorhabdus proteinsor fragments thereof, in particular novel insect inhibitory,bactericidal and fungicidal proteins.

The present invention provides an isolated nucleic acid moleculecomprising a nucleotide sequence, wherein: (1) the nucleotide sequencehybridizes under stringent conditions to a nucleotide sequence of asecond isolated nucleic acid molecule, wherein the hybridizing portionof the nucleotide sequence of the second isolated nucleic acid moleculeencodes a polypeptide or protein having an amino acid sequence selectedfrom the group consisting of SEQ ID NO:6109 to SEQ ID NO:12216; (2) thenucleotide sequence encodes a polypeptide or protein, wherein the aminoacid sequence of the polypeptide or protein is substantially identicalto any one set forth in SEQ ID NO:6109 to SEQ ID NO:12216; or (3) thenucleotide sequence is the complement of (1) or (2). In one embodiment,the nucleotide sequence is or is a portion of the isolated nucleic acidmolecule as disclosed herein, selected from the group consisting of SEQID NO:1 to SEQ ID NO:6108. The present invention provides an isolatedprotein having an amino acid sequence that is substantially identical toa member selected from group consisting of SEQ ID NO:6109 through SEQ IDNO:12216. By “substantially identical” or “substantial identity” as usedin reference to two amino acid sequences, it is meant that one aminoacid sequence is identical to the other amino acid sequence or has atleast 50% sequence identity, at least 70% sequence identity, preferablyat least 80%, more preferably at least 90%, and most preferably at least95% identity when compared to the other amino acid sequence as areference sequence using the programs described herein, preferably BLASTusing standard parameters, as described below. “Sequence identity” isdetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison and multiplying the result by 100 to yield the percentage ofsequence identity.

Polypeptides that are substantially similar share sequences in whichresidue positions are not identical and may differ by conservative aminoacid changes. Conservative amino acid substitutions refer to theinterchangeability of residues having similar side chains. “Conservativeamino acid substitutions” refer to substitutions of one or more aminoacids in a native amino acid sequence with another amino acid(s) havingsimilar side chains, resulting in a silent change. Conserved substitutesfor an amino acid within a native amino acid sequence can be selectedfrom other members of the group to which the naturally occurring aminoacid belongs.

Optimal alignment of sequences for comparison can use any means toanalyze sequence identity (homology) known in the art, e.g., by FGENESB(Softberry, Inc., Mount Kisco, N.Y.) that is based on Markov chainmodels of coding regions and translation and termination sites, by theBLAST algorithm (Altschul et al, J. Mol. Biol. 215: 403-410, 1990) thatis suitable for determining sequence similarity; by the progressivealignment method of termed “PILEUP” (Morrison, Mol. Biol. Evol.14:428-441, 1997); or by the local homology algorithm of Smith &Waterman (Adv. Appl. Math. 2: 482, 1981). One skilled in the art willrecognize that a value of sequence identity can be appropriatelyadjusted to determine corresponding sequence identity of two nucleotidesequences encoding the proteins of the present invention by taking intoaccount codon degeneracy, conservative amino acid substitutions, readingframe positioning and the like. Substantial identity of nucleotidesequences for these purposes normally means sequence identity of atleast 50%, preferably at least 60%, more preferably at least 70%, morepreferably at least 80%, more preferably at least 90%, and mostpreferably at least 95%. The present invention also includes an isolatednucleic acid comprising a nucleotide sequence encoding a polypeptidehaving an amino acid sequence set forth in any of SEQ ID NO:6109 to SEQID NO:12216 with conservative amino acid substitutions.

In a preferred embodiment of the present invention, the isolated nucleicacid molecule comprising a nucleotide sequence encodes an insectinhibitory protein. The nucleotide sequence encodes all or substantialportion of a polypeptide the amino acid sequence of which issubstantially identical to one of the sequences as set forth in SEQ IDNO's: 6903, 6904, 6905, 7110, 7179, 7514, 7776, 7777, 7803, 8275, 8277,8279, 8280, 8281, 8454, 8468, 8595, 9946, 10477, 10481, 10482, 10483,10484, 10485, 10486, 10487, 10488, 10551, 11147, 11688, 11690, 11691,11692 and 11693, wherein the nucleotide sequence is selected from thegroup consisting of SEQ ID NO's:795, 796, 797, 1002, 1071, 1406, 1668,1669, 1695, 2167, 2169, 2171, 2172, 2173, 2346, 2360, 2487, 3838, 4369,4373, 4374, 4375, 4376, 4377, 4378, 4379, 4380, 4443, 5039, 5580, 5582,5583, 5584 and 5585. The phrase “an insecticidal protein” or “an insectinhibitory protein” refers to any polypeptide or protein or asubstantial portion thereof that exhibits insect inhibitory activity,wherein the activity is manifested by inhibiting the growth ordevelopment of, or contributing substantially to, or causing the deathof a Coleopteran, a Dipteran, a Lepidopteran, a Hemipteran, aHymenopteran, or a sucking and piercing insect or insect larvae thereof.It also refers to any polypeptide or protein with modified amino acidsequence, such as sequence which has been mutated, truncated, increasedand the like and which maintains at least the insect inhibitory activityassociated with the native protein. Accordingly, the isolated nucleicacids encoding those polypeptide or protein with such modification arealso within the scope of the present invention.

The insect inhibitory proteins of the present invention may share somehomology to known insecticidal proteins. For instance, the polypeptidesequence as set forth in SEQ ID NO: 6903 exhibits 71% amino acidsequence homology to an insecticidal toxin complex protein TccB2 fromPhotorhabdus luminescens laumondii. The polypeptide sequence as setforth in SEQ ID NO: 6905 exhibits 61% amino acid sequence homology to aninsecticidal toxin complex protein TccA2 from Photorhabdus luminescenslaumondii. The polypeptide sequence as set forth in SEQ ID NO: 7110exhibits 57% amino acid sequence homology to an insecticidal toxincomplex protein TccC from Photorhabdus luminescens laumondii, etc.

In another aspect of the present invention, the isolated nucleic acidmolecule encodes all or a portion of a protein homologue to a knownprotein and may have important utility. For example, the isolatednucleic acid molecule encodes all or a portion of a hemolysin lipaseprotein homologue wherein the amino acid sequence of the proteinhomologue is substantially identical to one of the sequences as setforth in SEQ ID NO's: 6531, 6578, 6696, 7505, 7679, 7793, 7939, 8216,8220, 8222, 8366, 8745, 9199, 9212, 10143, 10306, 10325, 10683, 10919,10995, 10996, 11246, 11991 and 12000.

The isolated nucleic acid molecule comprising a nucleotide sequenceencodes all or a portion of a polyketide synthase homologue, wherein theamino acid sequence of the protein homologue is substantially identicalto one of the sequences as set forth in SEQ ID NO's: 7253, 8852, 8857,8864, 9558, 9560, 11014, 11583, 11587, 12090 and 12112. Polyketides aresmall bioactive molecules that are a class of small compounds linked bytheir biosynthetic pathways. The pathways and their products areparticularly abundant in soil microorganisms including Xenorhabdusnematophila. A large number of major pharmaceutical and agriculturalproducts have been derived from these complex natural products includinginsecticides, fungicides, antibacterials, anti-inflammatory,cancer-fighting agents, and cholesterol-lowering agents. Examples ofpolyketides include Rifamycins (Rifampin), Adriamycin (Doxorubicin),Erythromycin, Mevacor (Lovastatin), Ascomycin (Immunomycin), andSpinosad. Polyketides are produced by large proteins called synthases(or synthetases). There are an extraordinary number of polyketidessynthases from Xenorhabdus. In addition to polyketide synthasesXenorhabdus also contains an extraordinary number of related proteinscalled non-ribosomal peptide synthases (NRP synthase). These proteinsalso generate small molecules with a variety of biochemical functions.It is possible that any of these genes can be placed into the genome ofa plant and produces a substance (polyketide or non-ribosomal peptide)that can protect a plant against damage from insects, fungi, orbacteria. In addition, these genes can be placed in plants to generatepolyketides or non-ribosomal peptide for other uses includingpharmaceuticals.

The isolated nucleic acid molecule comprising a nucleotide sequenceencodes all or a portion of a protease homologue, wherein the amino acidsequence of the protein homologue is substantially identical to one ofthe sequences as set forth in SEQ ID NO's: 6308, 6309, 6310, 6393, 6537,6549, 6595, 6774, 6941, 6942, 7199, 7289, 7328, 7503, 7627, 7682, 7683,7749, 8152, 8300, 8301, 8870, 8957, 9108, 9263, 9265, 9296, 9319, 9343,9720, 9725, 9748, 9749, 9884, 10246, 10385, 10461, 10588, 10614, 10896,11020, 11830, 11831 and 11901. Protease plays very important roles in anorganism's metabolism and proteins synthesis and several types ofproteases have been reported. A processing protease is a protease thatcleaves a propeptide to generate a mature biochemically activepolypeptide (Enderlin and Ogrydziak, Yeast 10:67-79, 1994). Serineprotease is required for intramitochondrial proteolysis and maintenanceof respiratory function. Ubiquitin-specific protease (ubiquitinC-terminal hydrolase) of the 26S proteasome complex is involved invacuole biogenesis and osmoregulation. Inner membrane protease ofmitochondria acts in complex with IMP1P but has different substratespecificity for removal of signal peptidase serine protease of thesubtilisin family with broad proteolytic specificity (U.S. Pat. No.6,723,837). Therefore, the nucleotide sequences may find utility forthose skilled in the art in generating useful traits in plants or otherorganisms using available techniques.

The isolated nucleic acid molecule comprising a nucleotide sequenceencodes all or a portion of a chitinase homologue, wherein the aminoacid sequence of the protein homologue is substantially identical to oneof the sequences as set forth in SEQ ID NO's: 6902, 6906, 7325, 8047 and10542. The genes encoding these protein homologues may be overexpressedin plants as antifungal proteins to control fungal diseases in plants. Achitinase is one of several classes of antifungal proteins identifiedthat include chitinases, defensins, cysteine-rich chitin-bindingproteins, β-1,3-glucanases, permatins (including zeamatins), thionins,ribosome-inactivating proteins, and non-specific lipid transfer proteins(U.S. Pat. No. 6,573,361).

The isolated nucleic acid molecule comprising a nucleotide sequenceencodes all or a portion of a restriction enzyme homologue, wherein theamino acid sequence of the protein homologue is substantially identicalto one of the sequences as set forth in SEQ ID NO's: 8941, 8945, 9353,11041, 11042, 11113 and 11114. “Restriction enzyme” refers to an enzymethat recognizes a specific palindromic sequence of nucleotides in doublestranded DNA and cleaves both strands. Cleavage typically occurs withinthe restriction site. It is obvious to those skilled in the art aboutthe utility of these nucleotide sequences encoding the restrictionsenzyme homologues.

The isolated nucleic acid molecule comprising a nucleotide sequenceencodes all or a portion of a histone homologue, wherein the amino acidsequence of the protein homologue is substantially identical to one ofthe sequences as set forth in SEQ ID NO's: 6182, 7457, 7980, 8272, 8605,8765, 8778, 8861, 9590, 9802, 10293, 10449, 10469, 10762, 10812, 10926,11206, 11677 and 12135. Histones were not previously found in bacteria.However, histones are abundant and required for DNA organization in alleukaryotes. Genes with homology to histones and proteins that affecthistones, such histone deacetylases may affect histones in insects,disrupting normal cellular processes.

The isolated nucleic acid molecule comprising a nucleotide sequenceencodes all or a portion of a ferritin homologue the amino acid sequenceof which is substantially identical to one of the sequences as set forthin SEQ ID NO's: 6267, 6268 and 9272. These proteins may be used foroverexpression in plants may result in an increase of resistance toabiotic and biotic oxidative stresses. Overexpression of ferritinpromotes cellular productivity during limited water conditions toprevent formation of oxygen radicals (US Pat. Appl. Pub. No.20030233670).

The isolated nucleic acid molecule comprising a nucleotide sequenceencodes all or a portion of a drug metabolite transporter proteinhomologue, the amino acid sequence of which is substantially identicalto one of the sequences as set forth in SEQ ID NO's: 6836, 6837, 7447,8274, 8701, 9071, 9100, 9579, 10205, 10446, 10456, 10604, 10737 and11018.

The isolated nucleic acid molecule comprising a nucleotide sequenceencodes all or a portion of a protein homologue capable of conferringresistance to heavy metals, wherein the amino acid sequence of theprotein homologue is substantially identical to one of the sequences asset forth in SEQ ID NO's: 7432, 7741, 8701, 8706, 8828, 8829, 9071,9181, 9271, 10600 and 11018. Those genes may be introduced into cropplants to provide for resistance to a heavy metal in a poor growingconditions (U.S. Pat. No. 6,426,447). Therefore, those skilled in theart will find the utility of these nucleotide sequences in traitdevelopment in plants.

The isolated nucleic acid molecule comprising a nucleotide sequenceencodes all or a portion of a RTX (repeats in toxin) homologue, theamino acid sequence of which is substantially identical to one of thesequences as set forth in SEQ ID NO's: 9946, 10551 and 10643. RTXbelongs to the cytolytic toxin family. Within the medical sciences,there is growing interest in the potential utility of purified mammalianantibodies in the diagnosis and treatment of disease. For example,tagged antibodies directed against tumor cell surface antigens provide ahighly sensitive and specific means for detecting and classifyingvarious cancers. One therapeutic application using antibodies involvesthe administration of purified tumor-specific antibodies that arechemically coupled to cytotoxic agents. A class of cytotoxins that holdsparticular promise in the treatment of cancers consists of proteintoxins from plants. However, progress in the treatment of cancers usingchemically coupled antibodies and cytotoxins has been impeded by thelack of a cost effective means for producing these molecules in apharmaceutically acceptable grade and in commercially acceptablequantities. Therefore, the nucleotide sequences of the inventionencoding the cytotoxin homologs may be overexpressed in plants and largequantities of the cytotoxin proteins may be produced, isolated andpurified from the plants. These purified toxins may be used as atherapeutic agent (U.S. Pat. No. 6,140,075).

The isolated nucleic acid molecule comprising a nucleotide sequenceencodes all or a portion of a PapA protein homologue, the amino acidsequence of which is substantially identical to the sequence set forthin SEQ ID NO: 7514. PapA is shown to have cytotoxicity to larvalhemocyes of an insect and, therefore, may be used as an insecticidalprotein (Khandelwal et al., Biochem. Biophy. Res. Commun. 314: 943-949,2004).

The isolated nucleic acid molecule comprising a nucleotide sequenceencodes all or a portion of a pyoverdin protein homologue, the aminoacid sequence of which is substantially identical to the sequence setforth in SEQ ID NO: 6598. Pyoverdin is a fluorescent siderophoresecreted by Pseudomonas aeruginosa group. It's used to help the microbeleech iron out of its surroundings and is produced mostly in irondeficient environments. Pyoverdin competes directly with transferrin foriron and that it is an essential element for in vivo iron gathering andvirulence expression in P. aeruginosa. It is indicated that it playsessential role in bacterial growth and development (Meyer et al., InfectImmun. 64 (2): 518-523, 1996).

The isolated nucleic acid molecule comprising a nucleotide sequenceencodes all or a portion of a Zinc finger protein homologue, the aminoacid sequence of which is substantially identical to the sequence as setforth in SEQ ID NO: 8011. A zinc finger is part of a protein that canbind to DNA. Zinc-finger proteins regulate the expression of genes aswell as nucleic acid recognition, reverse transcription, signaltransduction and virus assembly and have prominent roles in many othercellular processes. Therefore, Zinc finger proteins and their metalbinding sites are promising targets for specific drug design to helpameliorate major diseases (Hanas et al., In: Zinc Finger proteins: fromatomic contact to cellular function, edited by Iuchi and Kuldell, 2004).

The isolated nucleic acid molecule comprising a nucleotide sequenceencodes all or a portion of an enterochelin protein homologue, the aminoacid sequence of which is substantially identical to the sequence as setforth in SEQ ID NO: 8528. Enterochelin proteins are iron-bindingcompound of E. coli and Salmonella spp. may have utilities inantibacterial arena (Antimicrob Agents Chemother. 18 (1): 63-68, 1980).

The isolated nucleic acid molecule comprising a nucleotide sequenceencodes all or a portion of a nonribosomal peptide homologue, the aminoacid sequence of which is substantially identical to one of thesequences as set forth in SEQ ID NO's: 6870, 7462, 7463, 8248, 8339,8492, 8530, 8546, 8547, 8856, 8860, 8863, 9225, 9226, 9227, 9552, 10073,10506, 10726, 11003, 11004, 11005, 11012, 11013, 11015, 11584, 11585,11588, 11590, 11591, 11844, 11845, 12085 and 12089.

The isolated nucleic acid molecule comprising a nucleotide sequenceencodes all or a portion of a protein homologous to a protein from aDrosophila species the amino acid sequence of which is substantiallyidentical to the sequence as set forth in SEQ ID NO: 7909. The geneencoding a protein homologous to a protein from Drosophila may affectpathogenesis in insects.

The present invention also relates to a class of isolated nucleic acidmolecules comprising promoter sequences or regulatory elements,particularly those found within SEQ ID NO:12217 through SEQ ID NO:14867or complements thereof. Particularly, the promoter sequence comprises anucleotide sequence, wherein: (1) the nucleotide sequence hybridizedunder stringent conditions to a nucleotide sequence of a second isolatednucleic acid molecule selected from the group consisting of SEQ IDNO:12217 through SEQ ID NO:14867 or complements thereof; (2) thenucleotide sequence is a portion of any sequence selected from the groupconsisting of SEQ ID NO:12217 through SEQ ID NO:14867; or (3) thenucleotide sequence is the complement of (1) or (2). As used herein, theterm “promoter” or “promoter sequence” means a nucleotide sequence thatis capable of, when located in cis to a structural nucleotide sequenceencoding a polypeptide or protein, functioning in a way that directsexpression of one or more mRNA molecules that encodes the polypeptide orprotein. Such promoter regions are typically found upstream of thetrinucleotide, ATG, at the start site of a polypeptide-coding region.Promoter molecules can also include DNA sequences from whichtranscription of tRNA or rRNA sequences are initiated.

The present invention also relates to an isolated nucleic acid moleculecomprising terminator sequences, particularly those found within SEQ IDNO:14868 through SEQ ID NO:16342 or complements thereof and refers to anucleotide sequence that is required for the termination reaction of thetranscription process. Termination involves recognition of the point atwhich no further bases should be added to a growing RNA chain. Toterminate transcription, the formation of phosphodiester bonds mustcease and the transcription complex must come apart. When the last baseis added to the RNA chain, the RNA-DNA hybrid is disrupted, the DNAreforms into a duplex state, and the RNA polymerase enzyme and RNAmolecule are both released from the DNA.

The present invention also relates to an isolated nucleic acid moleculethat encodes ribosomal RNA (rRNA), transfer RNA (tRNA) molecules.Particularly, the isolated nucleic acid molecule comprise a nucleotidesequence, wherein: (1) the nucleotide sequence hybridized understringent conditions to a nucleotide sequence of a second isolatednucleic acid molecule selected from the group consisting of SEQ IDNO:16343 through SEQ ID NO:16424 or complements thereof; (2) thenucleotide sequence is a portion of any sequence selected from the groupconsisting of SEQ ID NO:16343 through SEQ ID NO:16424; or (3) thenucleotide sequence is the complement of (1) or (2).

The isolated nucleic acid molecules of the present invention alsoinclude those comprising a substantial portion of a nucleotide sequenceselected from the group consisting of SEQ ID NO:1 through SEQ ID NO:6108or complements thereof. A “substantial portion” of a nucleotide sequencecomprises enough of the sequence to afford specific identificationand/or isolation of a nucleic acid fragment comprising the sequence. Ingeneral, gene specific oligonucleotide probes comprising 20-30contiguous nucleotides may be used in sequence-dependent methods of geneidentification (e.g., Southern hybridization) and isolation (e.g., insitu hybridization of bacterial colonies or bacteriophage plaques). Inaddition, short oligonucleotides of 12-15 bases may be used asamplification primers in PCR in order to obtain a particular nucleicacid fragment comprising the primers. The skilled artisan, having thebenefit of the sequences as reported herein, may now use all or asubstantial portion of the disclosed sequences for purposes known to himor her in this art. Accordingly, the present invention comprises thecomplete sequences as reported in the accompanying Sequence Listing, aswell as substantial portions of those sequences as defined above.

The nucleic acids of the present invention may be used to isolatenucleic acids encoding homologous proteins from the same or otherspecies, such as Photorhabdus, Serratia, Yersinia, Salmonella, E. coli,and Erwinia sp. Isolation of homologous genes using sequence-dependentprotocols is well known in the art. Examples of sequence-dependentprotocols include, but are not limited to, methods of nucleic acidhybridization, and methods of DNA and RNA amplification as exemplifiedby various uses of nucleic acid amplification technologies (e.g.,polymerase chain reaction, ligase chain reaction).

For example, genes encoding homologous proteins, either as cDNA's orgenomic DNA's, could be isolated directly by using all or a portion ofthe nucleic acids of the present invention as DNA hybridization probesto screen cDNA or genomic libraries from any desired organism employingmethodology well known to those skilled in the art. Methods for formingsuch libraries are well known in the art (Sambrook et al., MolecularCloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press, ColdSpring Harbor, 1989). Specific oligonucleotide probes based upon thenucleic acids of the present invention can be designed and synthesizedby methods known in the art. Moreover, the entire sequences of thenucleic acids can be used directly to synthesize DNA probes by methodsknown to the skilled artisan such as random primer DNA labeling, nicktranslation, or end-labeling techniques, or RNA probes using availablein vitro transcription systems. In addition, specific primers can bedesigned and used to amplify a part or all of the sequences. Theresulting amplification products can be labeled directly duringamplification reactions or labeled after amplification reactions, andused as probes to isolate full-length cDNA or genomic DNAs underconditions of appropriate stringency. Two short segments of the nucleicacids of the present invention may also be used in polymerase chainreaction protocols, for example, the RACE protocol (Frohman et al.,Proc. Natl. Acad. Sci. USA 85:8998, 1988), to amplify longer nucleicacids encoding homologous genes from DNA or RNA from other sources.

Nucleic acids of interest may also be synthesized, either completely orin part, especially where it is desirable to provide plant-preferredsequences, by well-known techniques as described in the technicalliterature. See, e.g., Carruthers et al. (Cold Spring Harbor Symp.Quant. Biol. 47:411-418, 1982) and Adams et al. (J. Am. Chem. Soc.105:661, 1983). Thus, all or a portion of the nucleic acids of thepresent invention may be synthesized using codons preferred by aselected plant host. Plant-preferred codons may be determined, forexample, from the codons used in the proteins expressed in a particularplant host species (Brown et al., U.S. Pat. No. 5,689,052). Othermodifications of the gene sequences may result in mutants havingslightly altered activity.

Availability of the nucleotide sequences encoding Xenorhabdus proteinsfacilitates immunological screening of DNA expression libraries.Synthetic polypeptides representing portions of the amino acid sequencesof Xenorhabdus proteins may be synthesized. These polypeptides can beused to immunize animals to produce polyclonal or monoclonal antibodieswith specificity for polypeptides or proteins comprising the amino acidsequences. These antibodies can be then be used to screen expressionlibraries to isolate genes of interest (Lerner, Adv. Immunol 36: 1,1984). It is understood that those skilled in the art are familiar withthe standard resource materials that describe specific conditions andprocedures for the construction, manipulation and isolation ofantibodies (see, for example, Harlow and Lane, In Antibodies: ALaboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.,1988).

The present invention relates to a method for obtaining a nucleic acidfrom a cell other than a Xenorhabdus Xs86068 cell comprising anucleotide sequence encoding a Xenorhabdus protein homologue the aminoacid sequence of which is at least 70% identical to a member selectedfrom the group consisting of SEQ ID NO:6109 to SEQ ID NO:12216. In apreferred embodiment, the method of the present invention for obtaininga nucleic acid encoding all or a substantial portion of the amino acidsequence of a Xenorhabdus protein homologue comprising: (a) probing anexpression library with a hybridization probe comprising a nucleotidesequence encoding a polypeptide having an amino acid sequence set forthin any of SEQ ID NO:6109 to SEQ ID NO:12216, or an amino acid sequenceset forth in any of SEQ ID NO:6109 to SEQ ID NO:12216 with conservativeamino acid substitutions; (b) identifying a DNA clone that hybridizes tothe hybridization probe; (c) isolating the DNA clone identified in step(b); and (d) sequencing the DNA fragment that comprises the cloneisolated in step (c) wherein the sequenced nucleic acid molecule encodesall or a substantial portion of the amino acid sequence of theXenorhabdus protein homologue.

In another preferred embodiment, the method of the present invention forobtaining a nucleic acid molecule from a cell other than a XenorhabdusXs86068 cell that encodes a substantial portion of an amino acidsequence of a Xenorhabdus protein homologue comprising: (a) synthesizinga first and a second oligonucleotide primers corresponding to a portionof the coding sequence of a second nucleic acid molecule set forth inSEQ ID NO:1 through SEQ ID NO:6108; and (b) amplifying a DNA insertpresent in a cloning vector using the first and second oligonucleotideprimers of step (a) wherein the amplified nucleic acid molecule encodesall or a substantial portion of the amino acid sequence of theXenorhabdus protein homologue.

The present invention, in another aspect, provides a substantiallypurified protein or polypeptide molecule comprising an amino acidsequence, wherein the amino acid sequence is defined as follows: (1) theamino acid sequence is encoded by a nucleotide sequence that is at least50% identical to all or a substantial portion of a coding sequencelocated within SEQ ID NO:1 through SEQ ID NO:6108; or (2) the amino acidsequence is substantially identical to a member selected from the groupconsisting of SEQ ID NO:6109 to SEQ ID NO:12216. In alternativeembodiments, the nucleotide sequence is at least 55% identical, at least60% identical, at least 65% identical, at least 70% identical, at least75% identical, at least 80% identical, at least 85% identical, at least90% identical, at least 95% identical to all or a substantial portion ofa coding sequence located within SEQ ID NO:1 through SEQ ID NO:6108. Ina further embodiment, the nucleotide sequence is 100% identical to allor a substantial portion of a coding sequence located within SEQ ID NO:1through SEQ ID NO:6108. In a still further embodiment, the amino acidsequence is at least 60% identical, at least 70% identical, at least 80%identical, at least 90% identical, at least 95% identical to a memberselected from the group consisting of SEQ ID NO:6109 to SEQ ID NO:12216.

The term “substantially purified protein or polypeptide molecule” refersto a protein sequence molecule separated from substantially all othermolecules normally associated with it in its native state. Morepreferably a substantially purified protein sequence molecule is thepredominant species present in a preparation. A substantially purifiedmolecule may be greater than 60% free, preferably 75% free, morepreferably 80% free, more preferably 90% free, and most preferably 95%free from the other molecules (exclusive of solvent) present in thenatural mixture.

It is well known in the art that proteins or polypeptides may undergomodifications, including post-translational modifications, such as, butnot limited to, disulfide bond formation, glycosylation,phosphorylation, or oligomerization. Thus, as used herein, the term“protein molecule” or “polypeptide molecule” includes any proteinmolecule that is modified by any biological or non-biological process.The terms “amino acid” and “amino acids” refer to all naturallyoccurring amino acids. This definition is meant to include norleucine,ornithine, homocysteine, and homoserine.

The polypeptides or proteins of the present invention may be producedvia chemical synthesis, or more preferably, by expression in a suitablebacterial or eukaryotic host. Suitable methods for expression of thepolypeptides or proteins are described by Sambrook et al. (ibid). Thepolypeptides or protein molecules of the present invention may alsoinclude fusion protein sequence molecules. A protein sequence moleculethat comprises one or more additional polypeptide regions not derivedfrom that protein molecule is a “fusion” protein sequence molecule. Suchmolecules may be derivatized to contain carbohydrate or other moieties(such as keyhole limpet hemocyanin, etc.). Fusion protein sequencemolecules of the present invention are preferably produced viarecombinant means.

Another aspect of the present invention concerns antibodies,single-chain antigen binding molecules, or other proteins thatspecifically bind to one or more of the protein sequence molecules ofthe present invention and their homologues, fusions or fragments. Suchantibodies may be used to quantitatively or qualitatively detect theprotein sequence molecules of the present invention. As used herein, anantibody or polypeptide is said to “specifically bind” to a proteinsequence molecule of the present invention if such binding is notcompetitively inhibited by the presence of non-related molecules. Forexample, the antibodies of the present invention bind to proteinsequence molecules of the present invention, in a more preferredembodiment, the antibodies of the present invention bind to proteinsequence molecules derived from Xenorhabdus that comprise SEQ ID NO:6109through SEQ ID NO:12216.

Nucleic acid molecules that encode all or part of the protein sequenceof the present invention can be expressed, via recombinant means, toyield protein or polypeptides that can in turn be used to elicitantibodies that are capable of binding the expressed protein orpolypeptide. Such antibodies may be used in immunoassays for thatprotein or polypeptide. Such protein or polypeptide-encoding molecules,or their fragments may be “fusion” molecules (i.e., a part of a largernucleic acid molecule) such that, upon expression, a fusion protein isproduced. It may be desirable to derivative the obtained antibodies, forexample, with a ligand group (such as biotin) or a detectable markergroup (such as a fluorescent group, a radioisotope or an enzyme). Suchantibodies may be used in immunoassays for that protein or may be usedto screen DNA expression libraries to isolate clones containingfull-length insert of genes (Lemer, Adv. Immunol. 36: 1, 1984).

In one embodiment, the antibodies of the present invention specificallybind to one or more of the insect inhibitory polypeptides or proteins ofthe present invention comprising SEQ ID NO's: 6903, 6904, 6905, 7110,7179, 7514, 7776, 7777, 7803, 8275, 8277, 8279, 8280, 8281, 8454, 8468,8595, 9946, 10477, 10481, 10482, 10483, 10484, 10485, 10486, 10487,10488, 10551, 11147, 11688, 11690, 11691, 11692 and 11693. Suchantibodies may be used to detect the presence of such insect inhibitorypolypeptides or proteins in a sample.

The present invention also provides a method for detecting an insectinhibitory polypeptide or protein in a biological sample, wherein themethod comprises the steps of: (1) obtaining a biological sample; (2)contacting the sample with an antibody that specifically binds to thepolypeptide or protein comprising SEQ ID NO: 6903, 6904, 6905, 7110,7179, 7514, 7776, 7777, 7803, 8275, 8277, 8279, 8280, 8281, 8454, 8468,8595, 9946, 10477, 10481, 10482, 10483, 10484, 10485, 10486, 10487,10488, 10551, 11147, 11688, 11690, 11691, 11692 or 11693, underconditions effective to allow the formation of complexes; and (3)detecting the complexes so formed.

The present invention also relates to a plant recombinant vector orconstruct comprising a structural nucleotide sequence encoding aXenorhabdus protein sequence comprising an amino acid sequence that isselected from the group consisting of SEQ ID NO:6108 through SEQ IDNO:12216. In one embodiment, a plant recombinant vector or construct ofthe present invention comprises a structural nucleotide sequenceencoding an insect inhibitory protein sequence of the present inventioncomprising an amino acid sequence that is selected from the groupconsisting of SEQ ID NO's: 6903, 6904, 6905, 7110, 7179, 7514, 7776,7777, 7803, 8275, 8277, 8279, 8280, 8281, 8454, 8468, 8595, 9946, 10477,10481, 10482, 10483, 10484, 10485, 10486, 10487, 10488, 10551, 11147,11688, 11690, 11691, 11692 and 11693. The present invention also relatesto a transformed plant cell or plant comprising in its genome anexogenous nucleic acid encoding one or more Xenorhabdus proteins orpolypeptides of the present invention. The present invention alsorelates to methods for creating a transgenic plant in which one or moreXenorhabdus proteins or polypeptides of the present invention areoverexpressed.

As used herein, “structural nucleotide sequence” refers to a nucleotidesequence that is expressed to produce a polypeptide. The term “genome”as it applies to plant cells encompasses not only chromosomal DNA foundwithin the nucleus, but organelle DNA found within subcellularcomponents of the cell. DNAs of the present invention introduced intoplant cells can therefore be either chromosomally integrated ororganelle-localized. The term “genome” as it applies to bacteriaencompasses both the chromosome and plasmids within a bacterial hostcell. Encoding DNAs of the present invention introduced into bacterialhost cells can therefore be either chromosomally integrated orplasmid-localized.

Methods that are well known to those skilled in the art may be used toconstruct the plant recombinant construct or vector of the presentinvention. These methods include in vitro recombinant DNA techniques,synthetic techniques, and in vivo genetic recombination. Such techniquesare described in Sambrook et al. (ibid); and Ausubel et al. (CurrentProtocols in Mol. Biol., John, Wiley & Sons, New York, N.Y., 1989).

A plant recombinant construct or vector of the present inventioncontains a structural nucleotide sequence encoding one or moreXenorhabdus proteins or polypeptides of the present invention as setforth in SEQ ID NO:6109 through SEQ ID NO:12216 and operably linkedregulatory sequences or control elements.

The term “operably linked”, as used in reference to a regulatorysequence and a structural nucleotide sequence, means that the regulatorysequence causes regulated expression of the operably linked structuralnucleotide sequence. “Regulatory sequences” or “control elements” referto nucleotide sequences located upstream (5′ noncoding sequences),within, or downstream (3′ non-translated sequences) of a structuralnucleotide sequence, and which influence the transcription, RNAprocessing or stability, or translation of the associated structuralnucleotide sequence. Regulatory sequences may include promoters,translation leader sequences, introns, and polyadenylation recognitionsequences.

It is understood by those skilled in the art that different promotersmay direct the expression of a gene in different tissues or cell types,or at different stages of development, or in response to differentenvironmental conditions. For example, promoters that may be used in thepresent invention include, but are not limited to, constitutivepromoters [e.g., the nopaline synthase (NOS) promoters (Ellis et al.,EMBO Journal 6:11-16, 1987); the cauliflower mosaic virus (CaMV) 35S(Fraley et al., U.S. Pat. No. 5,858,742); and actin promoters, such asthe Arabidopsis actin gene promoter (see, e.g., Huang, Plant Mol. Biol.33:125-139, 1997)], inducible promoter [e.g., the drought-induciblepromoter of maize (Busk, Plant J. 11:1285-1295, 1997; the cold, drought,and high salt inducible promoter from potato (Kirch, Plant Mol. Biol.33:897-909, 1997; and salicylic acid inducible promoter (Uknes et al.,Plant Cell 5:159-169, 1993)] and tissue-specific promoters [e.g.,leaf-specific promoters [e.g., Matsuoka, Plant J. 6:311-319, 1994;Shiina, Plant Physiol. 115-477-483, 1997); root-specific promoters(e.g., Samac et al., Plant Mol. Biol. 25: 587-596, 1994; Yamamoto, PlantCell 3:371-382, 1991), tuber-specific promoters (Hannapel, PlantPhysiol. 101: 703-704, 1993; Bevan et al., EMBO J. 8: 1899-1906, 1986),seed-specific promoters (e.g., Sheridan, Genetics 142:1009-1020, 1996;Abler, Plant Mol. Biol. 22:10131-1038, 1993) and pollen-specificpromoter (e.g., Guerrero, Mol. Gen. Genet. 224:161-168, 1990; Wakeley,Plant Mol. Biol. 37:187-192, 1992).

It is recognized that additional promoters that may be utilized aredescribed, for example, in U.S. Pat. Nos. 5,378,619, 5,391,725,5,428,147, 5,447,858, 5,608,144, 5,608,144, 5,614,399, 5,633,441,5,633,435, and 4,633,436. In addition, a tissue specific enhancer may beused (Fromm et al., The Plant Cell 1:977-984, 1989). It is furtherrecognized that since in most cases the exact boundaries of regulatorysequences have not been completely defined, DNA fragments of differentlengths may have identical promoter activity.

The “translation leader sequence” refers to a DNA sequence locatedbetween the promoter sequence of a gene and the coding sequence. Thetranslation leader sequence is present in the fully processed mRNAupstream of the translation start sequence. The translation leadersequence may affect processing of the primary transcript to mRNA, mRNAstability or translation efficiency. Examples of translation leadersequences include maize and petunia heat shock protein leaders (U.S.Pat. No. 5,362,865), plant virus coat protein leaders, and plant rubiscoleaders, among others (Turner and Foster, Molecular Biotechnology 3:225,1995).

The 3′ non-translated sequence or 3′ transcription termination regionmeans a DNA molecule linked to and located downstream of a structuralpolynucleotide molecule and includes polynucleotides that providepolyadenylation signal and other regulatory signals capable of affectingtranscription, mRNA processing or gene expression. The polyadenylationsignal functions in plants to cause the addition of polyadenylatenucleotides to the 3′ end of the mRNA precursor. The polyadenylationsequence can be derived from the natural gene, from a variety of plantgenes, or from T-DNA genes. An example of a 3′ transcription terminationregion is the nopaline synthase 3′ region (nos 3′; Fraley et al., Proc.Natl. Acad. Sci. USA, 80: 4803-4807, 1983). The use of different 3′nontranslated regions is exemplified by Ingelbrecht et al. (Plant Cell1:671-680, 1989).

A recombinant vector or construct of the present invention willtypically comprise a selectable marker that confers a selectablephenotype on plant cells. Selectable markers may also be used to selectfor plants or plant cells that contain the exogenous nucleic acidsencoding polypeptides or proteins of the present invention. The markermay encode biocide resistance, antibiotic resistance (e.g., kanamycin,G418 bleomycin, hygromycin, etc.), or herbicide resistance (e.g.,glyphosate, etc.). Examples of selectable markers include, but are notlimited to, a neo gene (Potrykus et al., Mol. Gen. Genet. 199:183-188,1985) which codes for kanamycin resistance and can be selected for usingkanamycin, G418, etc.; a mutant EPSP synthase gene (Hinchee et al.,Bio/Technology 6:915-922, 1988) which encodes glyphosate resistance; anda nitrilase gene which confers resistance to bromoxynil (Stalker et al.,J. Biol. Chem. 263:6310-6314, 1988).

A recombinant vector or construct of the present invention may alsoinclude a screenable marker. Screenable markers may be used to monitorexpression. Exemplary screenable markers include a β-glucuronidase oruidA gene (GUS) which encodes an enzyme for which various chromogenicsubstrates are known (Jefferson, Plant Mol. Biol, Rep. 5:387-405, 1987;Jefferson et al., EMBO J. 6:3901-3907, 1987); an R-locus gene(Dellaporta et al., Stadler Symposium 11:263-282, 1988); a β-lactamasegene (Sutcliffe et al., Proc. Ndtl. Acad. Sci. (U.S.A.) 75:3737-3741,1978); and a luciferase gene (Ow et al., Science 234:856-859, 1986).Included within the terms “selectable or screenable marker genes” arealso genes that encode a secretable marker whose secretion can bedetected as a means of identifying or selecting for transformed cells.Examples include markers that encode a secretable antigen that can beidentified by antibody interaction, or even secretable enzymes that canbe detected catalytically. Secretable proteins fall into a number ofclasses, including small, diffusible proteins detectable, e.g., byELISA, small active enzymes detectable in extracellular solution (e.g.,α-amylase, β-lactamase, phosphinothricin transferase), or proteins whichare inserted or trapped in the cell wall (such as proteins which includea leader sequence such as that found in the expression unit of extensionor tobacco PR-S). Other possible selectable and/or screenable markergenes will be apparent to those of skill in the art.

In preparing the DNA constructs of the present invention, the variouscomponents of the construct or fragments thereof will normally beinserted into a convenient cloning vector, e.g., a plasmid that iscapable of replication in a bacterial host, e.g., E. coli. Numerousvectors exist that have been described in the literature, many of whichare commercially available. After each cloning, the cloning vector withthe desired insert may be isolated and subjected to furthermanipulation, such as restriction digestion, insertion of new fragmentsor nucleotides, ligation, deletion, mutation, resection, etc. so as totailor the components of the desired sequence. Once the construct hasbeen completed, it may then be transferred to an appropriate vector forfurther manipulation in accordance with the manner of transformation ofthe host cell.

The present invention also provide a transgenic plant comprising in itsgenome an isolated nucleic acid which comprises: (1) a 5′ non-codingsequence which functions in the cell to cause the production of a mRNAmolecule; which is linked to (2) a structural nucleotide sequence,wherein the structural nucleotide sequence encodes a Xenorhabdus proteinsequence of the present invention that is substantially identical to amember selected from the group consisting of SEQ ID NO:6109 to SEQ IDNO:12216; which is linked to (3) a 3′ non-translated sequence thatfunctions in said cell to cause termination of transcription.

The term “transgenic plant” refers to a plant that contains an exogenousnucleic acid, which can be derived from the same plant species or from adifferent plant species. Transgenic plants of the present inventionpreferably have incorporated into their genome or transformed into theirchloroplast or plastid genomes a selected polynucleotide (or“transgene”), that comprises at least a structural nucleotide sequencethat encodes a polypeptide the amino acid sequence of which is selectedfrom the group consisting of SEQ ID NO:6109 to SEQ ID NO:12216 or, inparticular, an insect inhibitory polypeptide the amino acid sequence ofwhich is selected from the group consisting of SEQ ID NO's: 6903, 6904,6905, 7110, 7179, 7514, 7776, 7777, 7803, 8275, 8277, 8279, 8280, 8281,8454, 8468, 8595, 9946, 10477, 10481, 10482, 10483, 10484, 10485, 10486,10487, 10488, 10551, 11147, 11688, 11690, 11691, 11692 and 11693.Transgenic plants are also meant to comprise progeny (decendant,offspring, etc.) of any generation of such a transgenic plant. A seed ofany generation of all such transgenic insect-resistant plants whereinsaid seed comprises a DNA sequence encoding the polypeptide of thepresent invention is also an important aspect of the invention.

The DNA constructs of the present invention may be introduced into thegenome of a desired plant host by a variety of conventionaltransformation techniques, which are well known to those skilled in theart. Preferred methods of transformation of plant cells or tissues arethe Agrobacterium mediated transformation method and the biolistics orparticle-gun mediated transformation method. Suitable planttransformation vectors for the purpose of Agrobacterium mediatedtransformation include those derived from a Ti plasmid of Agrobacteriumtumefaciens, as well as those disclosed, e.g., by Herrera-Estrella etal. (Nature 303:209, 1983); Bevan (Nucleic Acids Res. 12: 8711-8721,1984); Klee et al. (Bio-Technology 3(7): 637-642, 1985); and EPOpublication 120,516. In addition to plant transformation vectors derivedfrom the Ti or root-inducing (Ri) plasmids of Agrobacterium, alternativemethods can be used to insert the DNA constructs of this invention intoplant cells. Such methods may involve, but are not limited to, forexample, the use of liposomes, electroporation, chemicals that increasefree DNA uptake, free DNA delivery via microprojectile bombardment, andtransformation using viruses or pollen.

A plasmid expression vector suitable for the introduction of a nucleicacid encoding a polypeptide or protein of the present invention inmonocots using electroporation or particle, gun mediated transformationis composed of the following: a promoter that is constitutive ortissue-specific; an intron that provides a splice site to facilitateexpression of the gene, such as the Hsp70 intron (PCT PublicationWO93/19189); and a 3′ polyadenylation sequence such as the nopalinesynthase 3′ sequence (NOS 3′; Fraley et al., Proc. Natl. Acad. Sci. USA80: 4803-4807, 1983). This expression cassette may be assembled on highcopy replicons suitable for the production of large quantities of DNA.

When adequate numbers of cells (or protoplasts) containing the exogenousnucleic acid encoding a polypeptide or protein of the present inventionare obtained, the cells (or protoplasts) are regenerated into wholeplants. Choice of methodology for the regeneration step is not critical,with suitable protocols being available for hosts from Leguminosae(alfalfa, soybean, clover, etc.), Umbelliferae (carrot, celery,parsnip), Cruciferae (cabbage, radish, canola/rapeseed, etc.),Cucurbitaceae (melons and cucumber), Gramineae (wheat, barley, rice,maize, etc.), Solanaceae (potato, tobacco, tomato, peppers), variousfloral crops, such as sunflower, and nut-bearing trees, such as almonds,cashews, walnuts, and pecans. See, for example, Ammirato et al.(Handbook of Plant Cell Culture-Crop Species. Macmillan Publ. Co.,1984); Shimamoto et al (Nature 338:274-276, 1989); Vasil et al.(Bio/Technology 8:429-434, 1990; Bio/Technology 10:667-674, 1992);Hayashimoto (Plant Physiol. 93:857-863, 1990); and Datta et al.(Bio-technology 8:736-740, 1990). Regeneration can also be obtained fromplant callus, explants, organs, or parts thereof. Such regenerationtechniques are described generally in Klee et al. (Ann. Rev. Plant Phys.38:467-486, 1987).

A transgenic plant formed using Agrobacterium transformation methodstypically contains a single exogenous gene on one chromosome. Suchtransgenic plants can be referred to as being heterozygous for the addedexogenous gene. More preferred is a transgenic plant that is homozygousfor the added exogenous gene; i.e., a transgenic plant that contains twoadded exogenous genes, one gene at the same locus on each chromosome ofa chromosome pair. A homozygous transgenic plant can be obtained bysexually mating (selfing) an independent segregant transgenic plant thatcontains a single exogenous gene, germinating some of the seeds producedand analyzing the resulting plants produced for the exogenous gene ofinterest.

The development or regeneration of transgenic plants containing theexogenous nucleic acid that encodes a polypeptide or protein of interestis well known in the art. Preferably, the regenerated plants areself-pollinated to provide homozygous transgenic plants, as discussedabove. Otherwise, pollen obtained from the regenerated plants is crossedto seed-grown plants of agronomically important lines. Conversely,pollen from plants of these important lines is used to pollinateregenerated plants. A transgenic plant of the present inventioncontaining a desired polypeptide or protein of the present invention iscultivated using methods well known to one skilled in the art.

Transgenic plants, that can be generated by practice of the presentinvention, include but are not limited to Acacia, alfalfa, aneth, apple,apricot, artichoke, arugula, asparagus, avocado, banana, barley, beans,beet, blackberry, blueberry, broccoli, brussels sprouts, cabbage,canola, cantaloupe, carrot, cassaya, cauliflower, celery, cherry,cilantro, citrus, clementines, coffee, corn, cotton, cucumber, Douglasfir, eggplant, endive, escarole, eucalyptus, fennel, figs, gourd, grape,grapefruit, honey dew, jicama, kiwifruit, lettuce, leeks, lemon, lime,pine, mango, melon, mushroom, nut, oat, okra, onion, orange, anornamental plant, papaya, parsley, pea, peach, peanut, pear, pepper,persimmon, pine, pineapple, plantain, plum, pomegranate, poplar, potato,pumpkin, quince, radiata pine, radicchio, radish, raspberry, rice, rye,sorghum, soybean, spinach, squash, strawberry, sugarbeet, sugarcane,sunflower, sweet potato, sweetgum, tangerine, tea, tobacco, tomato,turf, a vine, watermelon, wheat, yams, and zucchini.

The present invention also provides parts of the transgenic plants ofpresent invention. Plant parts, without limitation, include seed,endosperm, ovule and pollen. In a particularly preferred embodiment ofthe present invention, the plant part is a seed.

The present invention also further provides method for generating atransgenic plant comprising the steps of: a) introducing into the genomeof the plant an exogenous nucleic acid, wherein the exogenous nucleicacid comprises in the 5′ to 3′ direction i) a promoter that functions inthe cells of said plant, said promoter operably linked to; ii) astructural nucleic acid sequence encoding a polypeptide or protein ofthe present invention that is selected from the group consisting of SEQID NO:6109 to SEQ ID NO:12216 or, in particular, an insect inhibitorypolypeptide that is selected from the group consisting of SEQ ID NO's:6903, 6904, 6905, 7110, 7179, 7514, 7776, 7777, 7803, 8275, 8277, 8279,8280, 8281, 8454, 8468, 8595, 9946, 10477, 10481, 10482, 10483, 10484,10485, 10486, 10487, 10488, 10551, 11147, 11688, 11690, 11691, 11692 and11693, said structural nucleic acid sequence operably linked to; iii) a3′ non-translated nucleic acid sequence that functions in said cells ofsaid plant to cause transcriptional termination; b) obtainingtransformed plant cells containing the nucleic acid sequence of step(a); and c) regenerating from said transformed plant cells a transformedplant in which said polypeptide or protein is overexpressed.

Any of the isolated nucleic acid molecules of the present invention maybe introduced into a plant cell in a permanent or transient manner incombination with other genetic elements such as vectors, promoters,enhancers etc. Further any of the nucleic acid molecules encoding aXenorhabdus protein sequence of the present invention may be introducedinto a plant cell in a manner that allows for over expression of theprotein sequence encoded by the nucleic acid molecule.

The nucleotide sequences of the present invention may be introduced intoa wide variety of prokaryotic and eukaryotic microorganism hosts toexpress the Xenorhabdus polypeptide or protein of the present invention,particularly the insect inhibitory polypeptides or proteins of thepresent invention comprising an amino acid sequence that is selectedfrom the group consisting of SEQ ID NO's: 6903, 6904, 6905, 7110, 7179,7514, 7776, 7777, 7803, 8275, 8277, 8279, 8280, 8281, 8454, 8468, 8595,9946, 10477, 10481, 10482, 10483, 10484, 10485, 10486, 10487, 10488,10551, 11147, 11688, 11690, 11691, 11692 and 11693. The term“microorganism” includes prokaryotic and eukaryotic microbial speciessuch as bacteria and fungi. Illustrative prokaryotes, whetherGram-negative, Gram-positive, or otherwise, include Enterobacteriaceae,such as Escherichia, Erwinia, Shigella, Salmonella, and Proteus;Bacillaceae; Rhizobiceae, such as Rhizobium; Spirillaceae, such asphotobacterium, Zymomonas, Serratia, Aeromonas, Vibrio, Desulfovibrio,Spirillum; Lactobacillaceae; Pseudomonadaceae, such as Pseudomonas andAcetobacter; Azotobacteraceae, Actinomycetales, and Nitrobacteraceae.Among eukaryotes are fungi, such as Phycomycetes and Ascomycetes, whichincludes yeast, such as Saccharomyces and Schizosaccharomyces; andBasidiomycetes yeast, such as Rhodotorula, Aureobasidium,Sporobolomyces, and the like.

For the purpose of plant protection against insects, a large number ofmicroorganisms known to inhabit the phylloplane (the surface of theplant leaves) and/or the rhizosphere (the soil surrounding plant roots)of a wide variety of important crops may also be desirable host cellsfor manipulation, propagation, storage, delivery and/or mutagenesis ofthe disclosed recombinant constructs. These microorganisms includebacteria, algae, and fungi. Of particular interest are microorganisms,such as bacteria, e.g., genera Bacillus (including the species andsubspecies); Pseudomonas, Erwinia, Serratia, Klebsiella, Zanthomonas,Streptomyces, Rhizobium, Rhodopseudomonas, Methylophilius,Agrobacterium, Acetobacter, Lactobacillus, Arthrobacter, Azotobacter,Leuconostoc, and Alcaligenes; fungi, particularly yeast, e.g., generaSaccharomyces, Cryptococcus, Kluyveromyces, Sporobolomyces, Rhodotorula,and Aureobasidium.

The present invention also relates to a bacterial or a fungalrecombinant construct. The recombinant construct may comprise astructural nucleotide sequence encoding a Xenorhabdus protein sequencecomprising an amino acid sequence that is selected from the groupconsisting of SEQ ID NO:6109 to SEQ ID NO:12216. The present inventionalso relates to methods for obtaining a recombinant bacterial or fungalhost cell, comprising introducing into a bacterial or fungal host cellan exogenous nucleic acid molecule that is selected from the groupconsisting of SEQ ID NO:1 to SEQ ID NO:6108.

The recombinant construct for producing a polypeptide in a bacteriumalso contains an inducible promoter that is recognized by the hostbacterium and is operably linked to the nucleic acid encoding, forexample, the nucleic acid molecule encoding the Xenorhabdus proteinsequence of interest. Inducible promoters suitable for use withbacterial hosts include the β-lactamase, E. coli λ phage P_(L) andP_(R), and E. coli galactose, arabinose, alkaline phosphatase,tryptophan (trp), and lactose operon promoter systems and variationsthereof (Chang et al., Nature 275:615, 1978; Goeddel et al., Nature281:544, 1979; Guzman et al., J. Bacteriol. 174:7716-7728, 1992;Goeddel, Nucleic Acids Res. 8:4057, 1980; EP 36,776). Hybrid promoterssuch as the tac promoter (deBoer et al., Proc. Natl. Acad. Sci. (USA)80:21-25, 1983) and other known bacterial inducible promoters aresuitable (Siebenlist et al., Cell 20:269, 1980) may also be used.

The bacterial recombinant construct or vector may be a linear or aclosed circular plasmid. The vector system may be a single vector orplasmid or two or more vectors or plasmids that together contain thetotal DNA to be introduced into the genome of the bacterial host. Inaddition, the bacterial vector may be an expression vector. Nucleic acidmolecules encoding Xenorhabdus proteins or polypeptide can, for example,be suitably inserted into a replicable vector for expression in abacterium under the control of a suitable promoter for that bacterium.Many vectors are available for this purpose, and selection of theappropriate vector will depend mainly on the size of the nucleic acid tobe inserted into the vector and the particular host cell to betransformed with the vector. Each vector contains various componentsdepending on its function (amplification of DNA or expression of DNA)and the particular host cell with which it is compatible. The vectorcomponents for bacterial transformation generally include, but are notlimited to, one or more of the following: a signal sequence, an originof replication, one or more selectable marker genes, a promoter allowingthe expression of an exogenous nucleotide sequence and a structuralnucleotide sequence of the present invention.

In general, plasmid vectors containing replicon and control sequencesthat are derived from species compatible with the host cell are used inconnection with bacterial hosts. The vector ordinarily carries areplication site, as well as marking sequences that are capable ofproviding phenotypic selection in transformed cells. For example, E.coli is typically transformed using pBR322, a plasmid derived from an E.coli species (see, e.g., Bolivar et al., Gene 2:95, 1977). The pBR322plasmid contains genes for ampicillin and tetracycline resistance andthus provides easy means for identifying transformed cells. The pBR322plasmid, or other microbial plasmid or phage, also generally contains,or is modified to contain, promoters that can be used by the microbialorganism for expression of the selectable marker genes. In addition,nucleic acid molecules encoding Xenorhabdus proteins or polypeptides maybe expressed not only directly, but also as a fusion with anotherpolypeptide, preferably a signal sequence or other polypeptide having aspecific cleavage site at the N-terminus of the mature polypeptide. Thesuitable vectors containing one or more of the above-listed componentsmay be constructed employing standard recombinant DNA techniques.Isolated plasmids or DNA fragments are cleaved, tailored, and re-ligatedin the form desired to generate the plasmids required. Examples ofavailable bacterial expression vectors include, but are not limited to,the multifunctional E. Coli cloning and expression vectors such asBluescript™ (Stratagene, La Jolla, Calif.), in which, for example, aXenorhabdus protein sequence of the present invention, may be ligatedinto the vector in frame with sequences for the amino-terminal Met andthe subsequent 7 residues of β-galactosidase so that a hybrid protein isproduced; pIN vectors (Van Heeke and Schuster J. Biol. Chem.264:5503-5509, 1989); and the like. pGEX vectors (Promega, Madison,Wis.) may also be used to express foreign polypeptides as fusionproteins with glutathione S-transferase (GST). Bacterial cells used toproduce the polypeptide of interest for purposes of this invention arecultured in suitable media in which the promoters for the nucleic acidencoding the heterologous polypeptide can be artificially induced asdescribed generally, e.g., in Sambrook et al., ibid). Examples ofsuitable media are given in U.S. Pat. Nos. 5,304,472 and 5,342,763.

A yeast recombinant construct can typically include one or more of thefollowing: a promoter sequence, a fusion partner sequence, a leadersequence, a transcription termination sequence and a selectable marker.These elements can be combined into an expression cassette, which may bemaintained in a replicon, such as an extrachromosomal element (e.g.,plasmids) capable of stable maintenance in a host, such as yeast orbacteria. The replicon may have two replication systems, thus allowingit to be maintained, for example, in yeast for expression and in aprocaryotic host for cloning and amplification. Examples of suchyeast-bacteria shuttle vectors include YEp24 (Botstein et al., Gene,8:17-24, 1979), pC1/1 (Brake et al., Proc. Natl. Acad. Sci. USA,81:4642-4646, 1984), and YRp17 (Stinchcomb et al., J. Mol. Biol.,158:157, 1982).

The nucleotide sequence provided in one of SEQ ID NO:1 through SEQ IDNO:6108 or a fragment thereof, or a complement thereof, or a nucleotidesequence at least about 70% identical, preferably about 80% or about 90%identical, even more preferably about 95%, about 98% or 100% identicalto the nucleotide sequence provided in one of SEQ ID NO:1 through SEQ IDNO:6108 or a fragment thereof, or a complement thereof, can be“provided” in a variety of media to facilitate its use. Such a mediumcan also provide a subset thereof in a form that allows a skilledartisan to examine the sequences.

In one application of this embodiment, a nucleotide sequence of thepresent invention can be recorded on computer readable media. As usedherein, “computer readable media” refers to any medium that can be readand accessed directly by a computer. Such media include, but are notlimited to: magnetic storage media, such as floppy discs, hard disc,storage medium, and magnetic tape: optical storage media such as CD-ROM;electrical storage media such as RAM and ROM; and hybrids of thesecategories such as magnetic/optical storage media. A skilled artisan canreadily appreciate how any of the presently known computer readablemediums can be used to create a manufacture comprising computer readablemedium having recorded thereon a nucleotide sequence of the presentinvention.

By providing one or more of nucleotide sequences of the presentinvention, a skilled artisan can routinely access the sequenceinformation for a variety of purposes. Computer software is publiclyavailable which allows a skilled artisan to access sequence informationprovided in a computer readable medium. The examples which followdemonstrate how software which implements the BLAST (Altschul et al., J.Mol. Biol. 215:403-410, 1990) and BLAZE (Brutlag et al., Comp. Chem.17:203-207, 1993) search algorithms on a Sybase system can be used toidentify open reading frames (ORFs) within the genome that containhomology to ORFs or proteins from other organisms. Such ORFs are usefulin producing commercially important proteins such as enzymes used inamino acid biosynthesis, metabolism, transcription, translation, RNAprocessing, nucleic acid and a protein degradation, proteinmodification, and DNA replication, restriction, modification,recombination, and repair.

Nucleic acid molecules and fragments thereof of the present inventionmay be employed to obtain other nucleic acid molecules from the same orclosely related species. Such nucleic acid molecules include the nucleicacid molecules that encode the complete coding sequence of a protein,and promoters, and flanking sequences of such molecules. In addition,such nucleic acid molecules include nucleic acid molecules that encodefor other isozymes or gene family members. Such molecules can be readilyobtained by using the above-described nucleic acid molecules orfragments thereof to screen genomic libraries obtained from Xenorhabdus.Methods for forming such libraries are well known in the art.

Nucleic acid molecules and fragments thereof of the present inventionmay also be employed to obtain other nucleic acid molecules such asnucleic acid homologues. Such homologues include the nucleic acidhomologues of non-Xenorhabdus species including the nucleic acidmolecules that encode, in whole or in part, protein homologues of otherspecies or other organisms, sequences of genetic elements such aspromoters and transcriptional regulatory elements. Such molecules can bereadily obtained by using the above-described nucleic acid molecules orfragments thereof to screen cDNA or genomic libraries. Methods forforming such libraries are well known in the art. Such homologuemolecules may differ in their nucleotide sequences from those found inone or more of SEQ ID NO:1 through SEQ ID NO:6108 or complements thereofbecause complete complementarity is not needed for stable hybridization.The nucleic acid molecules of the present invention therefore alsoinclude molecules that, although capable of specifically hybridizingwith the nucleic acid molecules, may lack “complete complementarity.” Ina particular embodiment, methods for obtaining these molecules may beused [Frohman, M. A. et al., Proc. Natl. Acad. Sci. (U.S.A.)85:8998-9002, 1988; Ohara, O. et al., Proc. Natl. Acad. Sci. (U.S.A.)86:5673-5677, 1989].

The nucleic acid molecules of the present invention may be used forphysical mapping. Physical mapping, in conjunction with linkageanalysis, can enable the isolation of genes. Physical mapping has beenreported to identify the markers closest in terms of geneticrecombination to a gene target for cloning. Once a DNA marker is linkedto a gene of interest, the chromosome walking technique can be used tofind the genes via overlapping clones. For chromosome walking, randommolecular markers or established molecular linkage maps are used toconduct a search to localize the gene adjacent to one or more markers. Achromosome walk (Bukanov and Berg, Mo. Microbiol. 11:509-523, 1994;Birkenbihl and Vielnetter Nucleic Acids Res. 17:5057-5069, 1989; Wenzeland Herrmann, Nucleic Acids Res. 16:8323-8336, 1988) is then initiatedfrom the closest linked marker. Starting from the selected clones,labeled probes specific for the ends of the insert DNA are synthesizedand used as probes in hybridizations against a representative library.Clones hybridizing with one of the probes are picked and serve astemplates for the synthesis of new probes; by subsequent analysis,contigs are produced. The degree of overlap of the hybridizing clonesused to produce a contig can be determined by comparative restrictionanalysis. The most frequently used procedures are, fingerprinting(Coulson et al, Proc. Natl. Acad. Sci. (U.S.A.) 83:7821-7821, 1986;Knott et al., Nucleic Acids Res. 16:2601-2612, 1988; Eiglmeier et al.,Mol. Microbiol. 7:197-206, 1993), restriction fragment mapping (Smithand Birnstiel, Nucleic Acids Res. 3:2387-2398, 1976), or the“landmarking” technique (Charlebois et al. J. Mol. Biol. 222:509-524,1991).

Nucleic acid molecules of the present invention can be used to monitorexpression. A microarray-based method for high-throughput monitoring ofgene expression may be utilized to measure gene-specific hybridizationtargets. This ‘chip’-based approach involves using microarrays ofnucleic acid molecules as gene-specific hybridization targets toquantitatively measure expression of the corresponding genes (Schena etal., Science 270:467-470, 1995; Shalon, Ph.D. Thesis, StanfordUniversity, 1996). Every nucleotide in a large sequence can be queriedat the same time. Hybridization can be used to efficiently analyzenucleotide sequences.

It is understood that one or more of the molecules of the presentinvention, preferably one or more of the nucleic acid molecules orprotein molecules or fragments thereof of the present invention may beutilized in a microarray-based method. In one embodiment, the microarrayof the present invention comprises at least 10 nucleic acid molecules,more preferably at least 100 nucleic acid molecules, and even morepreferably at least 1000 nucleic acid molecules, that specificallyhybridize under stringent conditions to at least 10, at least 100, atleast 1000, nucleic acid molecules, respectively, encoding Xenorhabdusproteins or polypeptides or fragments thereof set forth in SEQ ID NO:1through SEQ ID NO:6108 or fragment thereof or complement. In a furtherembodiment, the microarray of the present invention comprises at least2,500 nucleic acid molecules that specifically hybridize under stringentconditions to at least 2,500 nucleic acid molecules that encode aXenorhabdus protein sequence or fragment thereof set forth in SEQ IDNO:6109 through SEQ ID NO: 12216. While it is understood that a singlenucleic acid molecule may encode more than one protein homologue orfragment thereof, in a preferred embodiment, at least 50%, preferably atleast 70%, more preferably at least 80%, even more preferably at least90% of the nucleic acid molecules that comprise the microarray containone protein or fragment thereof.

Nucleic acid molecules of the present invention may be used in sitedirected mutagenesis. Site-directed mutagenesis may be utilized tomodify nucleic acid sequences, particularly as it is a technique thatallows one or more of the amino acids encoded by a nucleic acid moleculeto be altered (e.g. a threonine to be replaced by a methionine). Threebasic methods for site-directed mutagenesis are often employed and theseare cassette mutagenesis (Wells et al., Gene 34:315-23, 1985), primerextension [Gilliam et al., Gene 12:129-137, 1980; Zoller and Smith,Methods Enzymol. 100:468-500, 1983; and Dalbadie-McFarland et al., Proc.Natl. Acad. Sci. (U.S.A.) 79:6409-6413, 1982] and methods based upon PCR(Scharf et al., Science 233:1076-1078, 1986); Higuchi et al., NucleicAcids Res. 16:7351-7367, 1988). Site-directed mutagenesis approaches arealso described in US Patent Pub. No. 20020151072, European Patent 0 385962, European Patent 0 359 472, and PCT Patent Application WO 93/07278.Any of the nucleic acid molecules of the present invention may either bemodified by site-directed mutagenesis or used as, for example, nucleicacid molecules that are used to target other nucleic acid molecules formodification. It is understood that mutants with more than one alterednucleotide can be constructed using techniques that practitionersskilled in the art are familiar with such as isolating restrictionfragments and ligating such fragments into an expression vector (see,for example, Sambrook et al., ibid).

Insect inhibitory protein-encoding nucleic acids of the presentinvention will find particular uses in the plant protection againstinsects. For instance, insect-resistant transgenic plants can begenerated by introducing the exogenous nucleic acids encoding an insectinhibitory polypeptide or protein or insect inhibitory fragment thereof,the amino acid sequence of which is substantially identical to asequence set forth in SEQ ID NO's: 6903, 6904, 6905, 7110, 7179, 7514,7776, 7777, 7803, 8275, 8277, 8279, 8280, 8281, 8454, 8468, 8595, 9946,10477, 10481, 10482, 10483, 10484, 10485, 10486, 10487, 10488, 10551,11147, 11688, 11690, 11691, 11692 and 11693. The methods for generatingsuch insect-resistant transgenic plants have been disclosed herein.

Insect inhibitory protein-encoding nucleic acids of the presentinvention will also find particular uses in engineering a transgenicmicroorganism (bacteria or fungi) to express the insect inhibitorypolypeptides or proteins of the present invention and then to apply themto the insect food source or allow them to reside in soil surroundingplant roots or on the surface of plant leaves. The transgenicmicroorganisms of the present invention may be used to produceXenorhabdus polypeptides or proteins of interest, particularly insectinhibitory polypeptides or proteins. Insect inhibitory polypeptides orproteins or insect inhibitory fragments thereof may be secreted, forexample as in bacterial systems, meaning targeted to either theperiplasm as for gram negative bacteria or localized to theextracellular space for gram negative or any other type of bacterium, orlocalized to the intracellular spaces within the cytoplasm. Suchcompositions may be administered to insects according to methods wellknown in the art.

Insecticidal proteins of the present invention can be used together withother insecticidal or pesticidal agents, such as organic chemicalcompositions, organo-phosphate compositions, nerve agents, diverseinsecticidal proteins such as Bt cry, vip, TIC, and EG relatedinsecticidal proteins, as well as with agents designed for dsRNAmediated gene suppression.

The principle object of the present invention is to provide a method foridentification of any gene or any protein encoded by any structural genecontained within a Xenorhabdus species, particularly those species whichare shown to exhibit the production of an insect inhibitory protein ormolecule or other similarly active composition, either alone or incombination with proteins or molecules or other similarly activecompositions which may be derived from the bacterium in its role as anatural symbiont within an insect pathogenic nematode host. Isolationand identification of a single insect pathogenic nematode speciesenables the skilled artisan to isolate at least one species ofXenorhabdus endosymbiotic bacteria from the haemolymph of an insectlarvae or adult which has been invaded by the isolated and identifiedhost nematode. The isolation and purification of an insect pathogenicnematode Xenorhabdus symbiont bacterium from an insect cadaver providesthe basis for obtaining an amount of genomic DNA from which a genomiclibrary can be constructed to represent the entire genome of thebacterial strain. The library can then be manipulated as describedherein to produce linear nucleotide sequences, which can then becompared to each other to identify regions of identity with which anoverlapping sequence can be generated to produce islands of linearsequence known as contigs because of the contiguous linear sequenceassembled from smaller bits of sequence data. The contigs can beassembled into a genomic map from which genes can be identified, andwherein translation of structural genes lead to further identificationof proteins having predicted structure and function based on homologiesof such predicted protein sequences as translated from open readingframes contained within the genome map, to proteins of known sequence,and perhaps also of known structure and function identified previouslyfrom other bacterial, viral, fungal, or other eukaryotic sources.

The Xenorhabdus strain Xs86068 and isolatable protein compositionsexhibiting insecticidal activity as disclosed herein will findparticular utility as insecticides for topical and/or systemicapplication to field crops, grasses, fruits and vegetables, andornamental plants. In a preferred embodiment, the bioinsecticidecomposition comprises an oil flowable suspension of bacterial cells thatexpresses a novel protein disclosed herein. In another importantembodiment, the bioinsecticide composition comprises a water dispersiblegranule. This granule comprises bacterial cells that express a novelinsecticidal protein disclosed herein. In a third important embodiment,the bioinsecticide composition comprises a wettable powder, dust,pellet, or colloidal concentrate. This powder comprises bacterial cellsthat express a novel insecticidal protein disclosed herein. Such dryforms of the insecticidal compositions may be formulated to dissolveimmediately upon wetting, or alternatively, dissolve in acontrolled-release, sustained-release, or other time-dependent manner.In a fourth important embodiment, the bioinsecticide compositioncomprises an aqueous suspension of bacterial cells such as thosedescribed above that express the insecticidal protein. Such aqueoussuspensions may be provided as a concentrated stock solution which isdiluted prior to application, or alternatively, as a diluted solution.

Exemplary bacterial cells for fulfilling the above methods may compriseXenorhabdus Xs86068 cells. However, bacteria such as Bacillus,Salmonella, Agrobacterium, Rhizobium, Erwinia, Azotobacter,Azospirillum, Klebsiella, Flavobacterium and Alcaligenes, otherXenorhabdus or Photorhabdus species, or Pseudomonas transformed with aDNA segment disclosed herein and expressing the insecticidal protein arealso contemplated to be useful.

Alternatively, the novel Xenorhabdus insecticidal proteins of thepresent invention may be prepared by native or recombinant bacterialexpression systems in vitro and isolated for subsequent fieldapplication. Such protein may be either in crude cell lysates,suspensions, colloids, etc., or alternatively may be purified, refined,buffered, and/or further processed, before formulating in an activebiocidal formulation. Likewise, under certain circumstances, it may bedesirable to isolate insecticidal proteins or whole cells from bacterialcultures expressing the insecticidal protein(s) of the present inventionand apply solutions, suspensions, or colloidal preparations of suchinsecticidal proteins or whole cells as the active bioinsecticidalcomposition.

Regardless of the method of application, the amount of the activecomponent(s) is applied at an insecticidally-effective amount, whichwill vary depending on such factors as, for example, the specificcoleopteran insects to be controlled, or the specific piercing andsucking insect to be controlled, the specific plant or crop to betreated, the environmental conditions, and the method, rate, andquantity of application of the insecticidally-active composition.

The insecticide compositions described herein may be made by formulatingeither the bacterial cells, insecticidal protein suspensions, orisolated protein components with the desired agriculturally acceptablecarrier (U.S. Pat. No. 6,177,615). The compositions may be formulatedprior to administration in an appropriate means such as lyophilized,freeze-dried, desiccated, or in an aqueous carrier, medium or suitablediluent, such as saline or other buffer. The formulated compositions maybe in the form of a dust or granular material, or a suspension in oil(vegetable or mineral), or water or oil/water emulsions, or as awettable powder, or in combination with any other carrier materialsuitable for agricultural application (U.S. Pat. Nos. 5,616,319 and5,942,658). Suitable agricultural carriers can be solid or liquid andare well known in the art. The insecticidal compositions of thisinvention are applied to the environment of the target coleopteran orpiercing and sucking insect, typically onto the foliage of the plant orcrop to be protected, by conventional methods, preferably by spraying(U.S. Pat. No. 6,177,615). The strength and duration of insecticidalapplication will be set with regard to conditions specific to theparticular pest(s), crop(s) to be treated and particular environmentalconditions. The proportional ratio of active ingredient to carrier willnaturally depend on the chemical nature, solubility, and stability ofthe insecticidal composition, as well as the particular formulationcontemplated.

Other application techniques, e.g., dusting, sprinkling, soaking, soilinjection, seed coating, seedling coating, spraying, aerating, misting,atomizing, and the like, are also feasible and may be required undercertain circumstances such as e.g., insects that cause root or stalkinfestation, or for application to delicate vegetation or ornamentalplants. These application procedures are also well known to those ofskill in the art.

The insecticidal compositions may be employed in the method of thepresent invention singly or in combination with other compounds,including and not limited to other insecticidal proteins and pesticides.The method of the invention may also be used in conjunction with othertreatments such as surfactants, detergents, polymers or time-releaseformulations. The insecticidal compositions of the present invention maybe formulated for either systemic or topical use.

Having now generally described the invention, the same will be morereadily understood through reference to the following examples that areprovided by way of illustration, and are not intended to be limiting ofthe present invention, unless specified.

EXAMPLES Example 1

This example illustrates the isolation of Xenorhabdus bacteria.

Xenorhabdus nematophila bacterium, strain Xs86068, was isolated fromentomopathogenic nematodes according to the following procedure.Entomopathogenic nematodes were isolated from soil samples andentomopathogenic nematode suspensions were prepared according to theentomopathogenic nematode baiting method as disclosed in the US patentapplication (application Ser. No. 09/897,516). A variety of fourthinstar insect larvae that included Western corn rootworm (WCR,Diabrotica virgifera virgifera), corn ear worm (CEW, Helicoverpa zea),tobacco bud worm (TBW, Heliothis virescens), black cut worm (BCW,Agrotis ipsilon), beet army worm (BAW, Spodoptera exigua), boll weevil(BWV, Anthomonas grandis grandis), and Galleria mellonella were placedindividually in a 24-well plate containing Whatman filters in each well.Approximately ten microliters (μL) of an entomopathogenic nematodesuspension were added into each well with one insect. The plates wassealed with Parafilm™ and placed at 25° C. in the dark. After 48 to 72hours dead insect larvae were removed from the 24-well plate. The insectlarvae were surface sterilized [20 milliliter (mL) H₂O, 3 mL 4M NaOH and1 mL 5% NaOCl) for 5 minutes and air-dried. The insect larvae were cutopen with sterile instruments on the lateral side without injuring thegut and the hemolymph was streaked on indicator plates (NBTA and NA).The agar plates were incubated at 30° C. in the dark for 48 hours.

Characteristic blue colonies were selected from the indicator plates:phase I Xenorhabdus bacteria were able to take up bromthymol blue dyefrom the NBT agar and form the blue colonies. Bacterial characterizationwas performed according to methods known to the one skilled in the art(Farmer, Bergey's Manual of Systematic Bacteriology, Vol. 1: 510-511,1984; Akhurst & Boemare, J. Gen. Microbiol., Vol. 133: 1835-1845, 1988;Boemare et al., Int. J. Syst. Bacteriol., Vol. 44: 249-255, 1993).

Single characteristic phase I colonies were picked up by an inoculationloop and suspended into BHI media (Brain Heart Infusion medium (Difco),32 g/l, 50 mL in a 250 mL baffled flask). The bacteria were grown at 25°C. at 280 rpm on a rotary shaker in the dark. After 24 hours 15%glycerol was added to the bacterial culture, 1.5 mL aliquots for stockcultures were placed into cryovials and stored at −80° C.

The isolated Xenorhabdus strain Xs86068 was deposited according to theBudapest Treaty on the International Recognition of the Deposit ofMicroorganisms for the Purpose of Patent Procedures with the AgricultureResearch Culture Collection (NRRL) International Depositary Authority at1815 North University Street, in Peoria, Ill., ZIP 61604, U.S.A., onJul. 26, 2004 and designated as NRRL B-30757. It is contemplated for useas a source for DNA sequences encoding insecticidal and other types ofuseful proteins, and when formulated into a composition of matter as aspray, powder or emulsion, for the treatment of plants or animals toinhibit insect infestation and the like.

Example 2

This example illustrates the construction and characterization of aXenorhabdus genomic library.

Genomic DNA from Xenorhabdus, strain Xs86068, was prepared forconstruction of a genomic library using methods well known in the art.Xs86068 bacterial cells were grown in brain heart infusion broth (Difco)for 42 hours at 25° C. to mid-exponential phase (OD650 approximately1.0). Cells were poured into ten 1.5 mL-microcentrifuge tubes and spunfor 5 minutes at about 10,000 RPM to pellet. The supernatant was removedand the cells were frozen. The frozen pellets were resuspended into 200μL of TE (10 mM Tris, 1 mM EDTA, pH 8.0). Genomic DNA was prepared fromthe frozen cell pellets using the Promega Genomic Preparation kitfollowing the instructions of the manufacturer (Promega Corp., Madison,Wis.). Ten DNA samples were prepared from the cells above, and two ofthe samples were resuspended into 50 μL of TE. Sample purity was testedand confirmed by digestion using the restriction enzymes EcoRI, HindIII,NotI, and SalI. The resuspended samples were used for the preparation ofthe genomic library.

The genomic library of Xenorhabdus strain Xs86068, LIB4695, was preparedaccording to standard procedures well known to those skilled in the art.Genomic DNA was sheared and then polished with T4 polymerase and T4polynucleotide kinase. LIB4695 was constructed from fragments 2-5 KB inlength. Size fractionated fragments were recovered from an agarose gel.Blunt end ligation was used to clone DNA fractions into the HincII siteof the standard cloning vector pUC18. The resulting ligation reactionswere transformed into E. coli DH10B. The resulting vector fragmentcontains an intact beta-lactamase coding sequence enabling selection oftransformed cells containing genomic DNA insertions on media containingampicillin. Ninety-six ampicillin resistant transformants wereinoculated into 96 well deep well boxes containing TB media andampicillin to determine the efficiency of the library construction. 95%of colonies arising from the transformation contained an insert,presumably derived from the genomic sequences. Approximately 15,000colony-forming units per microliter of ligation mix were obtained. Aboutthirty thousand individual recombinant colonies from each library wereselected for DNA sequence analysis of inserted genomic DNA.

Example 3

This example serves to illustrate the generation and assembly ofXenorhabdus genome sequences and the assembly into contiguous sequences.The two basic methods for the DNA sequencing are the chain terminationmethod of Sanger et al., Proc. Natl. Acad. Sci. (U.S.A) 74:5463-5467,1977) and the chemical degradation method of Maxam and Gilbert, Proc.Natl. Acad. Sci. (U.S.A) 74:560-564, 1977). PHRED (phragment editor,Phil Green, University of Washington) was used to call the bases fromthe sequence trace files and to assign quality scores to the bases.After the base calling was completed, sequence preprocessing wasperformed by removing 5′ and 3′ vector and linker sequences, accordingto standard procedures well known in the art. The preprocessed sequenceswere then assembled into contigs, or groups of overlapping sequences.Contigs were assembled using PHRAP (phragment assembly program, PhilGreen, University of Washington) using default assembly parameters.

A total of 494 contigs were obtained and contig sequences wererecognized as those sequences whose designations begin with Xb4695.C.All contig sequences were run through the annotation and gene selectionprocesses as described in Example 4 below. The contig sequences werelisted in the Sequence Listing file from SEQ ID NO:16425 to SEQ IDNO:16918.

Example 4

This example illustrates the identification and annotations of differentgenes within the 494 contigs assembled as described in Example 3.FGENESB gene prediction algorithm (Softberry, Inc., Mount Kisco, N.Y.)was used for this purpose. The genes and partial genes embedded in suchcontigs were identified through a series of bioinformatic analysesfollowing the instructions as described in the program. The X.nematophila genome from strain Xs86068, as assembled from LIB4695,consisted of 494 sequence contigs. The sequence contigs were annotatedto identify genes and gene regulation elements. As a result, 6108protein-coding genes (SEQ ID NO:1 through SEQ ID NO:6108), 2651promoters (SEQ ID NO:12217 through SEQ ID NO:14867), 1475 terminators(SEQ ID NO:14868 through SEQ ID NO:16342) and 82 ribosomal and transferRNA genes (SEQ ID NO:16343 through SEQ ID NO:16424) were identified.

The Xenorhabdus genome was annotated by searching for homology to genesof known functions. These searches were done using homology to wholeprotein using blast as well as similarity to protein domains using Pfamand Hidden Markov Model algorithms. The annotations were then associatedwith the genes. The genome annotation was completed with FGENESB, abacterial gene/operon prediction and annotation pipeline developed bySoftberry Inc. (Mount Kisco, N.Y., USA). The annotation database andparameters were updated and customized when processing X. nematophilagenome. These annotations were assembled into a database that could bequeried by searching for key words using wildcard text searches.

The analysis was done by performing keyword searches against theannotated genome sequences in the database. Since Xenorhabdus is aninsect pathogen, it may contain potent insecticidal molecules that aresimilar to the toxin complex (tc) toxins previously shown to beassociated with Photorhabdus. There may be many genes that areassociated with virulence and pathogenesis in other eukaryotes. Thesemay include, for example, hemolysins, lipases, and RTX (repeats intoxin) family of cytolytic toxins. Homologs of histone, proteinssequestering iron, polyketides and Non-ribosomal (NRP) peptides, etc.,were also searched for homology to genes of known functions. Theexemplary key words used to conduct the searches included tc, toxin,RTX, PapA, hemolysin, lipase, chitinase, protease, ferritin, iron,chelin, pyoverdin, resistance, restriction, insect, Drosophila, ketide,NRP, polyketides, non-ribosomal, polymer, nema and nematode, etc. A wildcard was used with all searches.

The search results have shown that the Xenorhabdus strain Xs86068 hasproteins that are homologous to many important known proteins orpolypeptides. The search for homologs has also led to some newdiscoveries. Discovery of histone homologs was unexpected as histoneswere not previously found in bacteria. These genes might make histonesthat would affect an insect's growth and development by disrupting itsnormal cellular processes. A PapA homolog was identified in thissequence pool. PapA has been demonstrated to have an insecticidalactivity (Khandelwal et al., Biochem. Biophy. Res. Commun. 314: 943-949,2004). The first step was done to look for any annotation containing theword “resistance” and put them into first class. Often these homologsreferred to resistance to metals (e.g. tellurite resistance) orantibiotics (e.g. tetracycline resistance). Resistance homologs may alsocame about from small phage-like particles called colicins. Theseproteins may often be evolved from phage tails. Polyketides andnon-ribosomal (NRP) peptides were very large proteins, often greaterthan 1000 amino acid residues. Proteins that affected fingi and insectskeletons included chitinases. Proteins sequestering iron were often avirulence determinant. Homologs identified included ferritin.

In summary, the nucleotide sequences identified in SEQ ID NO:1 throughtSEQ ID NO:12216 encode many useful Xenorhabdus polypeptides or proteins,including but not limited to insect inhibitory polypeptides or proteinsas set forth in SEQ ID NO's 6903, 6904, 6905, 7110, 7179, 7776, 7777,7803, 8275, 8277, 8279, 8280, 8281, 8454, 8468, 8595, 9946, 10477,10481, 10482, 10483, 10484, 10485, 10486, 10487, 10488, 10551, 11147,11688, 11690, 11691, 11692 and 11693; a PapA protein as set forth in SEQID NO:7514; hemolysin lipase protein homologues set forth in SEQ IDNO's: 6531, 6578, 6696, 7505, 7679, 7793, 7939, 8216, 8220, 8222, 8366,8745, 9199, 9212, 10143, 10306, 10325, 10683, 10919, 10995, 10996,11246, 11991 and 12000; polyketide synthases as set forth in SEQ IDNO's: 7253, 8852, 8857, 8864, 9558, 9560, 11014, 11583, 11587, 12090 and12112; protease homologs as set forth in SEQ ID NO's 6308, 6309, 6310,6393, 6537, 6549, 6595, 6774, 6941, 6942, 7199, 7289, 7328, 7503, 7627,7682, 7683, 7749, 8152, 8300, 8301, 8870, 8957, 9108, 9263, 9265, 9296,9319, 9343, 9720, 9725, 9748, 9749, 9884, 10246, 10385, 10461, 10588,10614, 10896, 11020, 11830, 11831 and 11901; chitinases as set forth inSEQ ID NO's 6902, 6906, 7325, 8047 and 10542; restriction enzymes as setforth in SEQ ID NO's 8941, 8945, 9353, 11041, 11042, 11113 and 11114;histone homologues as set forth in SEQ ID NO's 6182, 7457, 7980, 8272,8605, 8765, 8778, 8861, 9590, 9802, 10293, 10449, 10469, 10762, 10812,10926, 11206, 11677 and 12135; fenitin homologues as set forth in SEQ IDNO's 6267, 6268 and 9272; drug metabolite transporter protein homologuesset forth in SEQ ID NO's 6836, 6837, 7447, 8274, 8701, 9071, 9100, 9579,10205, 10446, 10456, 10604, 10737 and 11018; polypeptides or proteinscapable of conferring resistance to heavy metals or other toxiccompositions as set forth in SEQ ID NO's 7432, 7741, 8701, 8706, 8828,8829, 9071, 9181, 9271, 10600 and 11018; RTX (repeats in toxin)homologues as set forth in SEQ ID NO's: 9946, 10551 and 10643; apyoverdin protein homologue as set forth in SEQ ID NO: 6598; a Zincfinger protein homologue as set forth in SEQ ID NO: 8011; anenterochelin protein homologue as set forth in SEQ ID NO: 8528;nonribosomal peptide homologues as set forth in SEQ ID NO's: 6870, 7462,7463, 8248, 8339, 8492, 8530, 8546, 8547, 8856, 8860, 8863, 9225, 9226,9227, 9552, 10073, 10506, 10726, 11003, 11004, 11005, 11012, 11013,11015, 11584, 11585, 11588, 11590, 11591, 11844, 11845, 12085 and 12089;and a protein homologue to proteins from Drosophila species as set forthin SEQ ID NO: 7909. These proteins or polypeptides, offered by way ofillustration and not by way of limitation, are just some of theexemplary proteins or peptides from the Xenorhabdus strain Xs86068 thatare homologous to known proteins or polypeptides.

Example 5

Xenorhabdus strain Xs86068 was evaluated for its insecticidal activitiesusing the following procedure. Strain 86068 was evaluated with other twoXenorhabdus strains Xs86830 (isolated from Galleria melonella) andXs86832 (isolated from beet armyworm, BAW, Spodotera exigua) forcomparative purpose. Specifically, three 250 mL baffled flasks eachcontaining 50 mL BHI medium were each inoculated with 1.5 mL bacterialstock culture from each strain, respectively, and incubated at 25° C.and 280 rpm on a rotary shaker in the dark for 48 hours. The culturebroth was centrifuged at 2600×g for 30 minutes at 4° C. and decantedfrom the cell and debris pellet. The broth was then sterile-filtered(0.2 μm) and dialyzed. The culture supernatant was concentrated 5× andwas then used for bioassays to evaluate insect inhibitory properties.Five milliliter of the supernatant was applied to each insect larva forbioassay and the larvae were obtained using insect eggs obtained fromcommercial sources, hatched and reared using conventional methods knownin the art.

Insect inhibitory activities of these strains were evaluated againstmembers of the insects in the orders Coleoptera that included Westerncorn rootworm (WCR, Diabrotica virgifera virgifera) and cotton bollweevil (BWV, Anthomonas grandis grandis), Lepidoptera that includedtobacco budworm (TBW, Heliothis virescens), corn earworm (CEW,Helicoverpa zea), black cutworm (BCW, Agrotis ipsilon), and Hemipterathat included Western tarnished plant bug (WTPB, Lygus hespus). Insectinhibitory activity against Western corn rootworm larvae was evaluatedas follows. Xenorhabdus culture supernatant, control medium (BHI) orTris buffer, pH 7.0, was applied to the surface (about 0.38 cm²) of amodified artificial diet (Bioserv™; diet product F9757) in 20 μLaliquots. The plates were allowed to air-dry in a drying chamber (16-20°C.; 40-50% RH) and the wells were infested with single non-diapausingneonate WCR hatched from surface disinfested eggs (Pleau, Master ofScience Thesis, Saint Louis University, 1999). Plates were sealed,placed in a humidified growth chamber and maintained at 27° C. for theappropriate period (5-7 days). Mortality and stunting (0-3) scores werethen assessed and statistically analyzed (SAS institute, user's manualfor JMP version 3.2, 1989-1997). Twenty four insects per treatment wereused in all studies. Control mortality was generally less than 10%.

Insect inhibitory activity against Lepidopteran larvae was tested asfollows. Xenorhabdus culture supernatant, control medium (BHI) and Trisbuffer, pH 7.0, were applied directly to the surface (about 0.38 cm²) ofstandard artificial Lepidopteran diet (Southland Products Incorporated,Lake Village, AR; Lepidopteran multi-species diet) in 20 μL aliquots.The diet plates were allowed to air-dry in a drying chamber (16-20° C.;40-50% RH). The test wells were then infested with insect eggs of TBW,CEW or BCW suspended in agar. In the case of ECB, neonates were handinfested into the wells at one neonate per well. Following infestation,diet plates were sealed, placed in a humidity controlled growth chamberand maintained in the dark at 27° C. for the appropriate period of time.Mortality and stunting measurements were scored at day 5 andstatistically analyzed (SAS institute, 1989-1997, User's manual for JMPversion 3.2). Generally 24 insects per treatment were used in allstudies. Control mortality generally ranged from 0-12.5%.

Insect inhibitory activity against the cotton boll weevil was evaluatedas follows. Xenorhabdus supernatant, control medium (BHI) or Tris, pH7.0, were applied in 20 μL aliquots to the surface of 200 μL ofartificial diet (Bioserv™ Co., Frenchtown, N.J.; diet product F9247) andallowed to air-dry. Boll weevil eggs were then placed into the wells,the wells sealed and the plates held at 27° C., 60% relative humidity(RH) for 6 days. An activity score, based on confounding of grossproduction, growth and mortality, was then assessed and analyzedstatistically (SAS institute, ibid). Control mortality ranged between 0%and 25%.

Insect inhibitory activity was also tested against Lygus bugs (WesternTarnished Plant Bug (WTPB), Lygus hesperus Knight) in the orderHemiptera. The insect inhibitory activity against Lygus bug was testedas follows. Feeding domes were made using a dome-making machinemanufactured by Analytical Research Systems (Gainesville, Fla.).Briefly, the system used a vacuum to form domes from Parafilm™ sheetingusing an aluminum block template shaped in the form of a 96-wellmicrotiter-plate. To each such formed dome was added 40 uL of a 1:10(v/v) dilution of test solution in diet. The dome-molded Parafilm™ wasthen heat sealed with a sheet of Mylar. The resulting Parafilm™ domesheet (96-wells) was placed onto a 96-well flat-bottomed microtiterplate containing one Lygus nymph per well. The assay was typicallyscored after 4 days for mortality and stunting, using a scale of 0 (nomortality or stunting) to 3 (complete mortality).

The bioassay results demonstrated that the culture supernatantcontaining insecticidal proteins from X. nematophila, strain Xs86068,exhibited insecticidal activity against some of the insect speciestested. Specifically, the culture supernatant exhibited very stronginsecticidal activity against WCR, BWV and WTPB. All strains showed verystrong stunting effects against BWV and, among them, strain Xs86068showed the strongest stunting effect. Similarly, all strains showed highmortality rates and, among them, strain Xs86068 showed 100% mortalityrate. However, while other two strains demonstrated much lower mortalityeffects against WTPB, the strain Xs86068 gained the highest mortalityrate (90.5%). The overall insecticidal activity of strain Xs86068against BWV and WCR were comparable to the activities of strains Xs86830and Xs86832. Strain Xs86068 seemed to be more active against WTPB incomparison to strains Xs86830 and Xs86832. Therefore, the bioassaysusing the cultural supernatant exhibited insecticidal activities againstWCR, BWV and WTPB, but not against TBW, CEW and BCW.

In the other bioassay conducted, similar results were observed. Thestrain Xs86068 was tested against WCR and WTPB as 1× unprocessedsupernatant from a 48 h shaken culture (BHI medium, 25° C.). The culturewas used as un-heated, heated (boiled 20 min.), and concentrated 5-fold(3K MWCO). The untreated 1× culture was most active against WCR with100% mortality rate and the highest stunting effect rated as number 3.Similarly, the untreated 1× culture was most active against WTPB with90% mortality rate (stunting rate unavailable). Heating processdestroyed the insecticidal activity against WCR with 0% mortality ratebut only decreased the insecticidal activity against WTPB with 49%mortality. Concentration of the supernatant by 5-fold decreased theinsecticidal activity on both the insects with 12% mortality on WCR and82% mortality on WTPB. Therefore, the insecticidal activity against WCRand WTPB seems to be heat-labile.

The specification above describes exemplary embodiments of the presentinvention. It will be understood by those skilled in the art that,without departing from the scope and spirit of the present invention andwithout undue experimentation, the present invention can be performedwithin a wide range of equivalent parameters. While the presentinvention has been described in connection with specific embodiments, itwill be understood that it is capable of further modifications. Variouspermutations and combination of the elements provided in all the claimsthat follow are possible and fall within the scope of this invention.

SEQ ID NO's referred to herein are listed in the sequence listing onCD-ROM which accompanies this specification. All patent publicationsreferred to in this specification are incorporated herein by reference.

1. An isolated nucleotide molecule encoding an insect inhibitoryprotein, wherein said insect inhibitory protein comprises an amino acidsequence set forth as SEQ ID NO:7514.
 2. The isolated nucleotidemolecule of claim 1, wherein said insect inhibitory protein targets apest selected from the group consisting of a coleopteran insect pest, adipteran insect pest and a hemipteran insect pest.
 3. The isolatednucleotide molecule of claim 2, wherein said coleopteran insect pest isselected from the group consisting of cotton boll weevil (BWV,Anthomonas grandis grandis) and Western corn rootworm (WCR, Diabroticavirgfera Virgifera).
 4. The isolated nucleotide molecule of claim 2,wherein said hemipteran insect pest is a Western tarnished plant bug(WTPB, Lygus Hesperus).
 5. The isolated nucleotide molecule of claim 1that has the sequence set forth in SEQ ID NO:
 1406. 6. The isolatednucleotide molecule of claim 5, isolated from Xenorhabdus nematophilabacterium strain Xs86068, having an NRRL deposit number B-30757.
 7. Anisolated nucleotide molecule encoding an insect inhibitory protein,wherein said insect inhibitory protein comprises an amino acid sequenceat least 95% identical to SEQ ID NO:7514.
 8. A transgenic plantcomprising the nucleotide molecule of claim
 7. 9. An isolated nucleotidemolecule encoding an amino acid sequence Consistin of SEQ ID NO:7514.